WO2010019643A2 - Dispositif thérapeutique à base de cellules extracorporelles et système d'administration - Google Patents

Dispositif thérapeutique à base de cellules extracorporelles et système d'administration Download PDF

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Publication number
WO2010019643A2
WO2010019643A2 PCT/US2009/053516 US2009053516W WO2010019643A2 WO 2010019643 A2 WO2010019643 A2 WO 2010019643A2 US 2009053516 W US2009053516 W US 2009053516W WO 2010019643 A2 WO2010019643 A2 WO 2010019643A2
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WO
WIPO (PCT)
Prior art keywords
cell
cells
brecs
housing
substrate
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Application number
PCT/US2009/053516
Other languages
English (en)
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WO2010019643A3 (fr
WO2010019643A9 (fr
Inventor
H. David Humes
Deborah Buffington
Gretchen Hageman
Original Assignee
Innovative Biotherapies, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by Innovative Biotherapies, Inc. filed Critical Innovative Biotherapies, Inc.
Priority to EP09807211.9A priority Critical patent/EP2323725A4/fr
Priority to CA2734200A priority patent/CA2734200A1/fr
Publication of WO2010019643A2 publication Critical patent/WO2010019643A2/fr
Publication of WO2010019643A9 publication Critical patent/WO2010019643A9/fr
Publication of WO2010019643A3 publication Critical patent/WO2010019643A3/fr
Priority to US13/027,481 priority patent/US20110190679A1/en

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
    • A61M1/3687—Chemical treatment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/22—Urine; Urinary tract, e.g. kidney or bladder; Intraglomerular mesangial cells; Renal mesenchymal cells; Adrenal gland
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/34—Filtering material out of the blood by passing it through a membrane, i.e. hemofiltration or diafiltration
    • A61M1/3472—Filtering material out of the blood by passing it through a membrane, i.e. hemofiltration or diafiltration with treatment of the filtrate
    • A61M1/3486—Biological, chemical treatment, e.g. chemical precipitation; treatment by absorbents
    • A61M1/3489—Biological, chemical treatment, e.g. chemical precipitation; treatment by absorbents by biological cells, e.g. bioreactor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
    • A61M1/3687—Chemical treatment
    • A61M1/3689—Chemical treatment by biological cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells

Definitions

  • the present invention relates to an extracorporeal therapeutic device for delivering therapeutic molecules into a body. More particularly, this invention relates to an extracorporeal therapeutic device containing viable cells.
  • One way that the molecules can be delivered into a body is through blood circulation or other bodily fluids. When the device is introduced into the blood stream outside the body, the cells produce and secrete the therapeutic molecule into blood or fluid circulating past the device.
  • Drug delivery devices useful for introducing therapeutic molecules into a mammal have been the subject of considerable research.
  • the research has focused on the development of devices that deliver molecules produced from cellular metabolism.
  • Efforts have also focused on producing an implantable cell based delivery system that can remain in a patient for an extended period of time.
  • An implantable device for delivering a pre-selected molecule, for example, a hormone, into a mammal's systemic circulation is described in U.S. Pat. No. 6,716,208, the entire contents of which is incorporated by reference.
  • the device described comprises an implantable blood permeable element that can be anchored to an inner wall of an intact blood vessel and a capsule that is held in place within the blood vessel by the anchored blood permeable element.
  • the capsule encloses viable cells which produce and secrete the preselected molecule into blood passing the capsule.
  • the patent also describes a method for percutaneously introducing the device into a preselected blood vessel.
  • Intracorporeal cell based delivery devices must be sized to fit within a body, typically a body lumen (such as a blood vessel) and, accordingly, have certain size limitations because of the reduced- size requirements.
  • the anchors used to attach the device to the body e.g., blood vessel
  • the anchors used to attach the device to the body must be configured to secure the device without introducing trauma to the body.
  • implantable devices can be difficult to retrieve, especially if they are left within the body for an extended period of time.
  • the bioartificial kidney (BAK) set-up consists of a filtration unit (a conventional synthetic hemofiltration cartridge) followed in series by the tubule (RAD) unit.
  • the tubule unit is able to maintain viability because oxygen along with metabolic substrates and growth substances are delivered to the tubule cells from both intraluminal ultrafiltrate and blood in the extracapillary space. Immunoprotection of the cells is achieved due to the impenetrability of immunoglobulins and immunocompetent cells across the hollow fibers.
  • the present invention provides an extracorporeal cell based delivery system that is designed to address aspects of an intracorporeal cell based delivery system. Further, the present invention provides an extracorporeal device designed to introduce therapeutic agents into a mammal that secretes a pre-selected molecule or a combination of cell products directly into the blood stream or into a body fluid or body cavity and addresses the challenges of the prior art. The invention will be more clearly understood from the description, which follows.
  • a miniaturized cell therapy device will not require extensive extracorporeal blood pump systems.
  • a miniaturized device that could be stored at the clinical site for immediate use is required to succeed as a commercial product.
  • the current RAD is stored at a central manufacturing facility at 37 0 C and must be shipped at 37 0 C to the clinical site, delaying treatment and adding to the cost of therapy.
  • Development of a cell device that can be cryopreserved and stored at clinical sites can help safely bring the device to market.
  • the present invention provides an extracorporeal therapeutic device for delivery of a pre-selected molecule or cell products into a mammal, for example, into the circulatory system or body fluids of a mammal.
  • An embodiment of the invention enables molecules to be introduced into the circulatory system or a body cavity without invasive surgical procedures. Once the device is deployed, it delivers the molecule directly into the blood stream or body fluid.
  • the device of the invention is adapted to produce and thereafter secrete the pre-selected molecule or cell product into the blood stream or body fluid over a determined period of time.
  • the extracorporeal device and method provide an easy and reproducible system for delivering therapeutically effective amounts of a gene product, for example, a hormone, growth factor, anti-coagulant, immunomodulator, or the like, directly into the blood stream or body fluid of the recipient without the disadvantages of an invasive implantation procedure.
  • a gene product for example, a hormone, growth factor, anti-coagulant, immunomodulator, or the like
  • An extracorporeal device that administers a preselected molecule(s) into the mammal over a predetermined period presents advantages over the prior art.
  • An extracorporeal device has the advantage of being easily taken out of the circulation system compared to the efforts required to remove an implanted device.
  • the present invention provides an extracorporeal device for delivering, over a determined period of time, a preselected molecule or cell products into the systemic circulation of a mammal.
  • the present invention provides a method for non- surgically introducing the device into blood circulation of a mammal that is capable of delivering the preselected molecule or cell products into systemic circulation.
  • the device includes a capsule that contains viable cells which produce and secrete the preselected molecule into the blood stream.
  • the device may include an anchoring element, which anchors the device to an inner surface of a tube that circulates blood extracorporeally.
  • extracorporeal circuit as used in this specification embraces any tube or conduit outside the body that may be connected to the circulatory system or body fluid compartment in a mammal and provides for the flow of blood or fluid through the tube or conduit by natural (e.g., heart) or artificial (e.g., mechanical pump) circulation.
  • An extracorporeal device of the present invention is configured to be disposed in an extracorporeal circuit.
  • anchoring element as used in this specification embraces any structure that may be inserted into the lumen of an extracorporeal circulatory system blood tube or conduit and that, once inserted, may be anchored, for example, by hooks, barbs, or stents, to an inner surface of the tube or conduit.
  • the anchoring element may be a blood clot filter-type structure.
  • a variety of blood clot anti-migration filters useful in the practice of the invention are known in the art.
  • the currently preferred anchoring element is an anti-migration filter known as a "Greenfield® vena cava filter". Useful Greenfield® vena cava filters are described in detail in U.S. Pat. Nos.
  • capsule as used in this specification embraces any hollow structure dimensioned to fit within the lumen of a tube or conduit used in an extracorporeal circuit and does not occlude or prevent blood or fluid flow.
  • the capsule is held in place within the extracorporeal blood circuit by anchoring element(s).
  • the capsule may be retained upstream of the anchoring element, alternatively, the anchoring element may be located downstream of the anchoring element and retained in place by an attachment, for example, a hook or tether, extending from the anchoring element to the capsule.
  • the capsule may be conical or wedge-like in shape to decrease the turbulence of blood flowing past the capsule.
  • the capsule is formed from a material that can filter particles such that particles (including cells) below a certain size can pass through and particles above a certain size are prevented from passing through.
  • the filter forms an ultrafiltrate from the blood to minimize the entry of proteins greater than 100,000 molecular weight so that immunoglobulins can be excluded from the bathing media around the cells, especially for nonautologous cells not to activate an immunologic response.
  • the capsule may include either a single hollow fiber or a bundle of hollow fibers made from a semi-permeable membrane.
  • the semi-permeable membrane filter preferably has pores of a size sufficient to permit the diffusion of a preselected molecule or cell products therethrough but yet small enough to exclude the passage of cells therethrough.
  • the pores preferably are designed to permit the preselected molecule produced by the cells to diffuse directly into the blood stream passing the hollow fiber while preventing the cells from migrating out of the hollow fiber and into the systemic circulation.
  • a variety of polymers are useful in producing the biocompatible semi-permeable membrane of the present invention.
  • the viable cells enclosed within the semi-permeable hollow fiber(s) of the capsule preferably are eukaryotic cells, and most preferably are mammalian cells.
  • the device described herein may comprise cells which naturally produce and secrete the preselected molecule or other cell products, it is contemplated that genetically engineered cells, i.e., cells transfected with, and capable of expressing a nucleic acid encoding the pre-selected molecule, may likewise be used in the practice of the invention.
  • the preselected molecule can be a protein, and most preferably is a hormone, for example, erythropoietin or insulin. It is contemplated, however, that the device may be used to deliver into the systemic circulation any molecule that can be produced and secreted from a viable cell.
  • single cell types that produce and secrete a single preselected molecule may be used in the invention, it is understood that cells belonging to a particular cell type that produce and secrete a plurality of preselected molecules likewise may be used in the practice of the present invention. Similarly, it is contemplated that a plurality of cell types, wherein cells belonging to each cell type produce and secrete different preselected molecules, may be combined in a capsule thereby to produce a device that delivers a desirable combination of preselected molecules into the circulation.
  • Preferred embodiments of the device include three configurations. Each preferred configuration isolates the therapeutic cells to minimize the immune response.
  • a device in a first configuration, consists of a cartridge, a cell bearing unit which may be in the form of tubes attached to the cartridge and an anchoring system.
  • the therapeutic cells are disposed within the tubes and the cells are isolated by the size of the pores in the tube.
  • the cell bearing unit is in the form of disks that are disposed in the cartridge.
  • the therapeutic cells on the disks are protected from immunologic rejection by isolating the disks in the cartridge and providing pores in the cartridge that prevents the cells from being exposed to undesirable elements while allowing free physiologic exchange for the cells within the extracorporeal blood or fluid stream.
  • a third configuration is a combination of the configurations described above. Other configurations are possible.
  • fluids for treatment such as ultrafiltrate derived from blood or peritoneal fluid or peritoneal fluid itself, such that cells are protected from molecules in the body fluids having the ability to mount an immune response.
  • Such extracorporeal circuits can be used in various applications, including, for example, chronic applications and acute applications. In chronic applications, cells can be isolated from a biopsy taken from the subject to be treated and these cells can be expanded prior to use.
  • an immunoprotective barrier such as a filter
  • the filters may provide immunoprotection by limiting the immune- stimulating molecules from passing through and contacting the cells. Additionally, the animal is protected from the cells which could elicit an immune response by contact of the molecules produced by the cells.
  • Any cells can be used in embodiments according to the invention, such as eukaryotic cells, such as mammalian cells, and such as renal cells.
  • an extracorporeal therapeutic system includes a housing defining an interior space, a frozen substrate having a trabecular structure disposed within the housing, and at least one cell disposed on the substrate.
  • the housing can include an inlet for receiving a fluid and an outlet for releasing a processed fluid.
  • At least one flow separator can be disposed between the inlet and the substrate.
  • the substrate can be initially separate from the housing, and/or can be coated with a biomatrix material such as ⁇ collagen IV.
  • an extracorporeal therapeutic system in another aspect of the invention, includes a housing defining an interior space, a substrate including a carbon material coated with niobium disposed within the housing, and at least one cell disposed on the substrate.
  • the substrate can have a trabecular structure, and the substrate can be initially separate from the housing.
  • the substrate can be coated with collagen IV.
  • the substrate can also be coated with any three-dimensional biomatrix material that protects the integrity and durability of the carbon material, and the biomatrix material can promote or increase cell expansion, attachment, and/or viability.
  • the housing can include an inlet for receiving a fluid and an outlet for releasing a processed fluid.
  • an extracorporeal therapeutic system includes a housing, at least one hollow fiber associated with the housing, a biomatrix material disposed within the at least one hollow fiber, and at least one cell disposed within the biomatrix material. This aspect or any of the following aspects can have any of the following features.
  • the cell can be disposed upon a particle.
  • the biomatrix material can be selected from, but is not limited to, alginate solution, gelled alginate solution, or physiologic buffer, and the biomatrix material can also be selected from nutragen or matrigel.
  • Other examples of biomatrix material can include collagen I or any three-dimensional forming or formed biocompatible matrix.
  • the hollow fiber can be disposed within the housing, and the housing can include an inlet and an outlet.
  • a substrate for maintaining cells includes a carbon material coated with niobium and with collagen IV and has a trabecular structure. This aspect or any of the following aspects can have the following features.
  • the substrate can also include a cell that has the potential to provide therapeutic value, such as a eukaryotic cell, a mammalian cell, and/or a renal cell.
  • the substrate can be frozen.
  • a method for loading an extracorporeal therapeutic system includes thawing a cryopreserved cell and loading the cell into the system. This aspect or any of the following aspects can have any of the following features.
  • ean include a housing and at least one hollow fiber associated with the housing, and the cryopreserved cell is thawed, mixed with a biomatrix material, and loaded into the hollow fiber.
  • the system also or alternatively can include the cryopreserved cell disposed on a substrate, which can be loaded into the housing.
  • the substrate can include a carbon material coated with niobium and, optionally, collagen IV.
  • a method for extracorporeal therapy includes connecting an extracorporeal therapeutic system to the peritoneum of a mammal, so that a peritoneal fluid of the mammal circulates through the system.
  • the system can include a housing and at least one hollow fiber associated with the housing, and the system also can include a biomatrix material disposed within the hollow fiber and at least one cell disposed within the biomatrix material.
  • the system also or alternatively can include a housing and a substrate including a carbon material coated with niobium and, optionally, collagen IV, and at least one cell can be disposed on the substrate.
  • the system also or alternatively can include a housing and a frozen substrate having a trabeculated structure. At least one cell can be disposed on the substrate.
  • the housing can also include an inlet for receiving fluid and an outlet for releasing a processed fluid.
  • an extracorporeal cell based therapeutic device includes (a) an anchor system which can be capable of attaching the device to an extracorporeal tube, which when attached to the inner wall of the tube permits blood in the tube to pass therethrough; and (b) a capsule including a plurality of pores and having viable cells disposed therein, so that the capsule, when introduced into the tube, can be retained within the tube by the anchor system and the pores permit nutrients to enter the capsule to maintain viability of the cells disposed therein.
  • the capsule can be defined by a semi-permeable membrane, and the semi-permeable membrane can include a material selected from polyvinylidene fluoride, polyvinylchloride, polyurethane, polyalginate, polystyrene, polyurethane, polyvinyl alcohol, polyacrylonitrile, polyamide, polymethylmethacrylate, polyethylene oxide, polytetrafluorethylene, isocyanate, cellulose acetate, cellulose diacetate, cellulose triacetate, cellulose nitrate, polysulfone, and mixtures thereof.
  • the viable cells can be disposed on a plurality of filaments, and the filaments can be metallic.
  • the pores can be dimensioned to prevent passage of antibodies therethrough, and the cells can be disposed on at least one disk.
  • the cells can be eukaryotic cells, such as mammalian cells.
  • the capsule can be adapted to be separated from the tube by attaching and detaching the anchor system from the tube.
  • the capsule can include at least one hollow fiber.
  • the pores can permit solutes less than 150 kD to pass therethrough, and the device can be configured to provide a therapeutically significant amount of a molecule with or without using an artificial blood pump.
  • an extracorporeal cell based therapeutic device includes (a) an attachment system anchorable to an inner wall of an extracorporeal tube adapted to provide a conduit for blood, which when anchored to the inner wall of a tube permits blood to circulate; and (b) a capsule defining a plurality of pores and having viable cells disposed therein, so that the capsule can be retained within the tube by the attachment system and the pores permit nutrients to enter the capsule to maintain viability of the cells disposed therein.
  • the pores can be dimensioned to exclude an agent in the blood which is detrimental to cell viability.
  • the anchor can be metallic.
  • the capsule when introduced into the tube can be retained upstream of the attachment system, and the capsule can include at least one disk.
  • the cells can also be genetically engineered cells.
  • a method of introducing an extracorporeal cell- based therapeutic device into the circulatory system of a mammal includes the steps of: (a) anchoring an attachment system to an inner wall of an extracorporeal tube adapted to provide a conduit for bodily fluid, which when anchored to the inner wall of a tube permits the bodily fluid to circulate; and (b) inserting a capsule defining a plurality of pores and having viable cells disposed therein, so that the capsule can retained within the tube by the attachment system and the pores permit nutrients to enter the capsule to maintain viability of the cells disposed therein.
  • This aspect or any of the following aspects can have any of the following features.
  • the attachment system can be introduced into the tube via a catheter
  • the capsule can be introduced into the tube via a catheter.
  • the device can be used to deliver a therapeutic amount of a molecule in the conduit with or without the use of an artificial body fluid pump.
  • the conduit can be attached to the peritoneum and the peritoneal fluid passes through the conduit.
  • an extracorporeal cell based therapeutic device includes (a) an attachment system anchorable to an inner wall of an extracorporeal tube adapted to provide a conduit for a bodily fluid, which when anchored to the inner wall of a tube permits the bodily fluid to circulate; and (b) a capsule defining a plurality of pores and having viable cells disposed therein, so that the capsule, when introduced into the tube, can be retained within the tube by the attachment system and the pores permit nutrients to enter the capsule to maintain viability of the cells disposed therein.
  • the tube can be adapted to be connected to the peritoneum and the bodily fluid includes peritoneal fluid, and the tube can be adapted to be connected to blood vessels and the bodily fluid includes blood.
  • the attachment system can be separable from the tube.
  • Figure 1 is a perspective side view of a device according to an embodiment of the present invention.
  • Figure 2 is a perspective side view of a device according to another embodiment of the present invention.
  • Figure 3 is a detail cross section of the downstream end of the device illustrated in
  • Figure 4 is a cross section of the device illustrated in Figure 2 and shows the flow of fluid through the device;
  • Figure 5 is a perspective view of another embodiment of the invention.
  • Figure 6 is a schematic cross section illustrating the embodiment of Figure 5;
  • Figure 7 is a detail view of an ultrafiltrate tube illustrated in Figure 5 and 6;
  • Figure 8 is a schematic view of an extracorporeal blood circuit that can be used with embodiments of the present invention.
  • Figures 9-12 are graphs representing testing of embodiments of the present invention.
  • Figure 13 is a perspective side view of another embodiment of the present invention and certain components of that embodiment
  • Figure 14 is a perspective side view of the device illustrated in Figure 13;
  • Figure 15 is a perspective view of another embodiment of the present invention.
  • Figure 16 is an exploded view of the embodiment illustrated in Figure 15;
  • Figure 17 A is a schematic view of a hollow fiber extracorporeal treatment device according to the invention and
  • Figure 17B is a schematic depicting cell seeding of the embodiment;
  • Figures 18A and B are schematic views of two extracorporeal blood circuits that can be used with an embodiment of the present invention, such as those shown in Figures 13-
  • Figure 19 is a schematic view of an extracorporeal peritoneal fluid circuit that can be used with an embodiment of the present invention, such as those shown in Figures 13-16;
  • Figure 20 is schematic view of another extracorporeal peritoneal fluid circuit that can be used with an embodiment of the present invention, such as those shown in Figures 13-
  • Figure 21 is a schematic view of another extracorporeal peritoneal fluid circuit that can be used with an embodiment of the present invention, such as those shown in Figures 13-
  • Figure 22 is a schematic of an extracorporeal blood circuit that can be used with an embodiment of the present invention, such as that shown in Figures 17A and B;
  • Figure 23 is a graph of the effect of renal assist device intervention on mean arterial pressure in a septic pig
  • Figure 24 is a graph of the effect of renal assist device intervention on cardiac output in a septic pig
  • Figure 25 is a graph of the effect of renal assist device intervention on stroke volume in a septic pig
  • Figure 26 is a graph of the effect of renal assist device intervention on hematocrit in a septic pig;
  • Figure 27 is a graph of the effect of renal assist device intervention on the survival rate of a septic pig;
  • Figure 28 is a graph of the number of primary porcine renal epithelial cells
  • Figures 29 A-D show cell growth on carbon disks;
  • Figure 30 is a graph of the average oxygen consumption for pre- and post- cryopreserved PPREC cells;
  • Figure 31 is a graph of glucose consumption rates for pre- and post-cryopreserved
  • Figure 32 is a graph of lactate production for pre- and post-cryopreserved PPREC cells;
  • Figure 33 is a graph of the survival times of a septic pig treated with a renal assist device, a sham renal assist device, and embodiments of the present invention (the device of
  • Figure 34 is a graph of non- stimulated and Lipopolysaccharide (LPS)-stimulated normal leukocytes under standard media and PPREC-conditioned media exposure;
  • Figure 35 is a graph of an assessment of leukocyte activation in multiple cell type populations under normal conditions, after 5 hours of sepsis and after maximal stimulations by phorbol myristate acetate (PMA);
  • PMA phorbol myristate acetate
  • Figure 36 is a graph of a Kaplan-Meier survival curve through 180 days comparing continuous venovenous hemofiltration (CVVH) therapy and CVVH therapy plus RAD therapy;
  • Figure 37 is a graph of lamb-renal epithelial cell (LREC) active disk oxygen consumption rates versus fixed disk oxygen consumption rates based on in vitro testing of the embodiment illustrated in Figure 13;
  • LREC lamb-renal epithelial cell
  • Figure 38 is a graph of the difference in glucose consumption rates of LRECs after a freeze thaw process based on in vitro testing of the embodiment illustrated in Figure 13;
  • Figure 39 is a graph of the difference in oxygen consumption rates of LRECs after a freeze thaw process based on in vitro testing of the embodiment illustrated in Figure 13;
  • Figure 40 is a graph of the LREC oxygen consumption rates determined by ex vivo testing using the circuits illustrated in Figures 19-20;
  • Figure 41 is a graph of the oxygen consumption rates determined by ex vivo testing using the circuits illustrated in Figures 19-20;
  • Figure 42 is a graph of the pH change in peritoneal dialysate (PD) solution and serum, as assessed via arterial blood gas analysis, after recycling commences in testing using the circuits illustrated in Figures 19-20;
  • Figure 43 is a graph of the bicarbonate change in PD solution and serum, as assessed via arterial blood gas analysis, after recycling commences in testing using the circuits illustrated in Figures 19-20;
  • Figure 44 is a graph of the potassium change in PD solution and serum, as assessed via arterial blood gas analysis, after recycling commences in testing using the circuits illustrated in Figures 19-20;
  • Figure 45 is a graph of the calcium change in PD solution and serum, as assessed via an Abbott Point-of-Care i-STAT analyzer, after recycling commences in testing of the circuits illustrated in Figures 19-20;
  • Figure 46 is a graph of lactate production by live cells of the embodiment illustrated in Figures 15 and 16;
  • Figure 47 is a graph of glucose consumption pre- and post-cryopreservation during testing of the embodiment illustrated in Figures 15 and 16;
  • Figure 48 is an image of HEP-G2 cells grown to near tissue density in an embodiment of the present invention.
  • Figure 49 is a graph of the growth curve of two flasks with PPREC- seeded microcarriers;
  • Figure 50 is a graph of lactate production of PPREC-seeded microcarriers;
  • Figure 51 is a graph of oxygen consumption of PPREC-seeded microcarriers
  • Figure 52 shows histology images of hollow fiber loaded PPREC-seeded microcarriers taken after 24 hours in culture.
  • the image on the left is a 5X magnification of microcarriers loaded in a 500 ⁇ m inner diameter fiber.
  • the image on the right is at 1OX magnification;
  • Figures 53A-D show images of DAPI staining of PPRECs on gelatin microcarriers at 5X magnification.
  • Figure 53A shows an image of microcarriers pre-freeze.
  • Figure 53B shows an image of microcarriers post-thaw that were frozen in CryostorTM CS5 cryopreservative medium.
  • Figure 53C shows an image of microcarriers post-thaw frozen in 5% DMSO.
  • Figure 53D shows an image of microcarriers post-thaw frozen in 10% DMSO; and
  • Figure 54 is a graph of a comparison of IL-8 secretion rates on baseline and LPS- stimulated conditions in PPREC in two-dimensional culture.
  • Figure 55 is a schematic view of another extracorporeal peritoneal fluid circuit that can be used with an embodiment of the present invention, such as those shown in Figures 13-
  • FIG. 56 is a graph of CDl Ib mean fluorescent intensity (MFI) during the course of human renal epithelial cell (HREC)-seeded BRECS-d cellular therapy in uremic sheep.
  • MFI was measured at the following time points: (1) PRESURDl, which was 1 day prior to sheep nephrectomy; (2) PREBRDO, which was the time point just prior to BRECS-d therapy when the sheep were at their most uremic state; (3) BRDl to BRD5, which corresponded to 1- 5 days of BRECS-d therapy, respectively; (4) BR FINAL, which was the final day of BRECS- d therapy, in this case day 6, when the BRECS-d unit was removed; (5) POSTBRDl, which was 24 hours following BRECS-d removal; and (6) FINAL, which was the last time point of this study, occurring at 48 hours following BRECS-d removal.
  • MFI levels as shown are average values calculated based on the number of animals that remained in the study
  • Figure 57 is a graph of the average oxygen consumption for LREC-seeded BRECS- d units and HREC-seeded BRECS-d units during the course of cellular therapy in normal and uremic sheep, respectively.
  • the BRECS-d units were maintained for up to 13 days of therapy in normal sheep and up to 6 days of therapy in uremic sheep.
  • Figures 58A-D are images of HRECs grown on collagen IV-coated carbon disks from BRECS-d units. Photographs are from three-dimensional microscopic fields of cut carbon disks to show en face labeling of surface antigens.
  • Figure 58 A is an image of DAPI staining.
  • Figure 58B is an overlay immunohistochemistry (IHC) image of ZO-I and AT-I staining, indicative of renal epithelial cell tight junctions and centralized cilia, respectively.
  • IHC immunohistochemistry
  • FIG 58C is an IHC image of CD 13, which is a brush border enzyme that facilitates Na +2 dependent amino acid transport.
  • Figure 58D is an IHC image of ⁇ -glutamyltranspeptidase ( ⁇ GT), which is a brush border enzyme that facilitates glutathione metabolism. All primary antibodies were visualized with either the secondary antibody anti-rabbit IgG AlexaFluor 488 conjugate or anti-mouse IgG AlexaFluor 594 conjugate.
  • Figure 58A was taken with a 5X objective.
  • Figures 58B-D were taken with a 1OX objective.
  • Figure 59 is a table showing commercially available antibodies that were used in the immunohistochemical analysis of HRECs.
  • GSH glutathione
  • Figure 62 is graph showing temperature changes in four acellular BRECS-d units thawed over time using the thawing protocol described in Example 8. The four studies demonstrate internal temperature control without reaching temperatures above 39°C.
  • the average cell number per BRECS-d unit at various time points in culture is shown (Pre-Freeze and Post- Thaw represent before freezing and post thawing, respectively).
  • Three point moving averages (value at the designated time point averaged with the 2 successive preceding time point values) were generated to display an accurate trendline.
  • LqN Pre-FREEZE” and "LqN Post-THAW” are data from before freezing and storage in liquid nitrogen at -140 0 C and after thawing, respectively;
  • "-80 Pre-FREEZE” and "-80 Post-THAW” are data from before freezing at - 140 0 C and storage at - 80 0 C and after thawing, respectively).
  • the average cell number shown represents the average value calculated from a data set at each time point that included those units stored at -80 0 C for 1 week and those units stored at -80 0 C for 1 month.
  • Points A and B denote cell numbers before freezing and after thawing, respectively, for units transferred from liquid nitrogen storage to -80 0 C.
  • Points C and D show cell numbers before freezing and after thawing, respectively, for units stored only in liquid nitrogen vapor phase.
  • Three point moving averages (value at the designated time point averaged with the 2 successive preceding time point values) were generated to display an accurate trendline.
  • the x-axis reflects actual days in culture and does not include days in cryopreservation.
  • Figure 65 is a graph showing temperature change as recorded by three thermocouples (TCl, TC3, TC4) placed inside a cryopreserved aceullular BRECS-d unit initially at the liquid nitrogen vapor phase (-140 0 C). The unit was transferred to a Styrofoam cooler with dry ice, sealed and monitored over 36 hours to determine the length of time temperature could be maintained.
  • the present invention provides an extracorporeal device for delivering molecules into a mammal, for example, the systemic circulation of a mammal.
  • the device of the invention is adapted for introduction into an extracorporeal circuit, such as into an extracorporeal blood conduit or a peritoneal fluid circuit. After introduction into the extracorporeal circuit, such as the extracorporeal blood conduit, the device permits the preselected molecule(s) or cell product(s) to diffuse out of the device.
  • these pre-selected molecule(s) or cell product(s) diffuse out of the device and into the blood stream or body cavity of the recipient, which in certain aspects does so in response to blood parameters, for example, oxygen tension in the case of erythropoietin- producing cells. Also, the glucose concentrations in the bathing media around the insulin producing cells can stimulate the production of insulin. [0098]
  • the embodiments of the invention have at least some of the following advantages. Because the device is extracorporeal, there is less size constraint and the cell bearing units can be sized larger than if a device was implanted. Further, because the device is not implanted in the body, the anchoring system does not need to be designed to be atraumatic.
  • the device includes a capsule or housing that holds cell-bearing material.
  • the cells on the cell bearing material are prevented from provoking an immunological response by isolating the cells using a porous material that creates a barrier between the blood and the cells.
  • the barrier allows the cells to have sufficient physiological exchange (e.g., drawing sustaining nutrients and oxygen) with the blood and delivery of the metabolic products produced by the cells.
  • the metabolic products may be in response to small messenger molecules that might be circulated in the blood stream as a result of the patient's pathologic condition.
  • the metabolic production can be stimulated by introducing a messenger molecule into the bloodstream of the patient.
  • cells in devices according to the invention are also spared from opportunities of clotting.
  • the primary concern of clotting is alleviated by removing contact of the device with blood.
  • Additional concerns related to blood viscosity, nutrient concentration, and immunological response are also alleviated by allowing an altered bodily fluid, such as ultrafiltrate derived from blood or peritoneal fluid, or with peritoneal fluid itself, to contact the cells.
  • the cells may draw nutrients and oxygen and deliver their metabolic products to these fluids.
  • an autologous cell source can be utilized.
  • filters placed pre- and post-device in an extracorporeal circuit optionally may or may not be used.
  • An embodiment of the device of the present invention includes an anchor system that secures the device to an inner wall of a extracorporeal blood circuit.
  • the anchor system may have arms that are adapted to be in a reduced profile configuration during delivery into the blood circuit and expand into a delivered profile so that the arms expand and extend to the blood conduit.
  • the anchoring system may use hooks, barbs, or stents disposed upon the arm of the anchor system.
  • the anchoring system is designed such that when anchored to the wall of the blood conduit, the system permits blood in the vessel to pass through and around the device.
  • the device includes a shape, e.g., cone, facing upstream that minimizes turbulence of blood as it flows beyond the capsule.
  • the device comprises a semipermeable housing containing viable cells which produce and secrete the pre- selected molecule.
  • the viable cells may be disposed on a flat surface such as a disk.
  • the cells are contained within porous tubes that allow the physiologic activity of the cells to occur and from which the preselected molecule or cell products can flow into the bloodstream or body fluids. The tubes may extend beyond the capsule.
  • Figure 1 illustrates schematically a device 10 useful in the practice of an embodiment of the present invention.
  • the device 10 includes a nose cone 12 and a body 14 that is used to secure a cell bearing unit 16 comprised of hollow fibers 18 made of a semi-permeable material which encloses viable cells for delivering the preselected molecules.
  • the nose cone and the body of the device support the cell bearing unit 16 comprising the hollow fibers 18.
  • the viable cells may be attached to an inner surface of a fiber. Whether the cells are attached to the inner surface will depend upon the cell type included in the device. For example, some cell types grow in an anchorage dependent manner upon a solid surface while other cell types have no anchorage dependency and grow in suspension. The choice of cell type, however, is dependent upon the desired application.
  • the device of Figure 1 is attached to an extracorporeal blood circuit by a suitable anchor system 20 that includes anchors 22.
  • Two anchors 22 are illustrated, however, more or fewer than two could be used in a suitable design.
  • the anchors are configured to hold the device securely in the extracorporeal conduit (shown in Figure 8).
  • the anchors may be retractable in a reduced profile for delivery and expanded into a deployed profile when the device is at the appropriate location on the conduit.
  • Various springy or resilient biocompatible material may be used including nitinol or other spring material.
  • Figures 2-4 illustrate another embodiment of the invention where the device 30 includes a nose cone 32 and a body 34 into which the cell bearing unit 36 is attached.
  • the cell bearing unit includes several semi-permeable membrane disks 38 which contain the viable cells.
  • the size and number of disks used may vary depending on the type and amount of the preselected molecule being delivered. It is contemplated that semipermeable membranes may be defined by either the same or different polymeric compositions.
  • An anchoring system 40 with anchors 42 is structured similarly and operates in a similar manner as the anchoring system described in connection with Figure 1. [0106] A detail view of the disk 38 is illustrated in Figure 3.
  • the disk 38 is approx 1 cm in diameter and has, in one embodiment, cells associated with the membranes such that the cells are exposed to the blood or fluid constituents for nutrients and the cells are able to deliver the desired product(s) for the blood.
  • the membranes have a trabecular structure to enhance the exchange of physiologic material.
  • the disk is similar to a coin that is trabeculated and porous. The configuration allows flow-through of various media components and anchorage dependent growth especially of epithelial cells at higher tissue density.
  • Figure 4 illustrates a device in a blood conduit. Blood flow is illustrated by the wavy lines.
  • a filter 44 is provided in the device to exclude large blood constituents, e.g., macrophages and immunoglobulins, from causing an undesired immune response with the cells on the disks 38.
  • the filtered material 46 also called ultrafiltrate, is allowed to flow across the disks 38. In other embodiments, they may flow through the membranes.
  • the ultrafiltrate provides sustaining nutrients to the cells on the disk and carries away the products of cell metabolism.
  • the resultant stream of ultrafiltrate blood and the products of metabolism is schematically illustrated as wavy dotted lines 48.
  • a filter can be used at the downstream outlet of the device.
  • the hollow tubes may be constructed to provide a multi-layered structure.
  • the device 50 includes a porous nose cone 52 that filters out large blood constituents and a body 54 that holds one end of a series of hollow tubes 56.
  • a single hollow tube is identified by reference numeral 58.
  • the body includes a manifold (illustrated in Figure 6) that distributes the filtered blood into the center of the hollow tubes, one cross section of which is illustrated in Figure 7.
  • the distal end of the hollow tubes may be blocked (not illustrated) to provide desirable flow dynamics or to prevent the flow of unfiltered blood from being introduced to the tubes. As such, the flow path of the filtered blood can be through the tube or through the wall, depending on the desired construct.
  • the hollow tube 58 includes a central lumen 62 through which the filtered blood passes.
  • Cells 64 are disposed along a tubular medium (illustrated in axial cross section) between an inner wall 66 and an outer wall 68 and cells are disposed such that filtered blood or fluid travels across the cells. The cells are thus provided with life sustaining nutrients enabling the production of desirable cell products.
  • the outer wall of the hollow tube 58 allows the cellular products to be delivered through the tube but does not allow large particles into the tube to contact the cells. Both the inner and outer wall may have such filtering function.
  • the end of the tube is not blocked and the filtrate is allowed to bath the cells and has a sufficient flow rate such that the cells are provided with the proper amount of nutrients and the unfiltered blood is not allowed to travel upstream to the cells.
  • anchoring systems that may be used in the instant invention include devices that provide blood clot filtering or blood vessel stents.
  • Useful anchoring elements are characterized by their ability to be anchored within the lumen of a conduit without occluding or preventing blood flow.
  • One of the advantages of the present invention is that the extracorporeal device anchoring system does not have to be designed to be secured in a blood vessel without damaging the blood vessel. This minimizes the concern for tissue damage.
  • Blood clot filters are used routinely by medical practitioners to prevent the migration of potentially life threatening blood clots within the vasculature.
  • Blood clot filters typically are designed to be implanted and anchored within the lumen of a blood vessel. When implanted, the anti-migration filters permit blood in the vessel to pass while simultaneously trapping blood clots.
  • the devices and techniques useful toward attaching a filter to a blood vessel can be applied to extracorporeal devices. Of course, because the device is not intended to be introduced into the vasculature, the design of the anchors need not have the high level of precision required for their use.
  • Greenfield® filters 4,817,600 and 5,059,205, referred to in the art as Greenfield® filters and available from Medi-Tech®, Boston Scientific Corporation, Natick, Mass., are particularly well suited to the practice of the invention.
  • the cone-shaped Greenfield® vena cava filters are designed to provide maximal entrapment area for trapping blood clots while maintaining patency of the blood vessel after trapping emboli.
  • the spacing between the six legs of the Greenfield® vena cava filters ensures the trapping of emboli greater than 3 mm (Greenfield et al (1989) "Venous Interruption" Chapter 68, pp.
  • the filters may be able to capture capsules greater than 3 mm in diameter.
  • the device is incorporated into a tube that is inserted into an extracorporeal conduit. In this embodiment, the anchors are unnecessary because the device is secured within the tube.
  • Device Design [0115] The extracorporeal drug delivery device of the various embodiments of the present invention may be capable of delivering a preselected drug or cell products over an extended period of time.
  • the size of the capsule can be scaled for higher delivery rates by increasing the size of the capsule and the drug delivery media.
  • the device permits delivery of the preselected molecule over defined periods of time
  • another important consideration in the design of the device is the configuration that will maintain the viability of the cells enclosed in the device. It is understood that a variety of factors, for example: the supply of oxygen and nutrients to the cells in the capsule; the removal of waste products from the cells in the capsule; the minimization of host immune responses directed against the cells in the capsule; the proliferative activity of the cells; and whether cells located at the center of the capsules are susceptible to pressure necrosis, all of which may influence the design and preparation of a cell containing tube(s).
  • the hollow fibers preferably are produced from a semi-permeable membrane having pores dimensioned to permit the diffusion of oxygen and nutrients into the lumen of the hollow fiber while permitting the efflux of cellular waste products and the pre- selected molecule out of the hollow fiber.
  • the pores preferably are dimensioned to exclude the passage of cells therethrough. Accordingly, the pores are designed to prevent migration of the viable cells from the lumen of the hollow fiber into the blood steam, thereby maintaining the implanted cells at a single location in the host to facilitate their subsequent removal if or when necessary.
  • the pores also are designed to prevent the influx of the hosts immune cells, for example, macrophages and lymphocytes, which if allowed to enter the lumen of the hollow fibers may be detrimental to the viability of the cells enclosed therein.
  • the membrane therefore, provides an immuno-protected environment that protects cells enclosed therein from an immune response. This may be an important consideration if the implanted cells are non- autologous in nature. If autologous cells are used per size restrictions longer than molecular dimension then the design would be modified accordingly.
  • the hollow fibers comprising, or for incorporation within, the capsule may be produced from biocompatible polymers which include, but are not limited to, polyvinylchloride, polyvinylidene fluoride, polyurethane isocyanate, polyalginate, cellulose acetate, cellulose diacetate, cellulose triacetate, cellulose nitrate, polysulfone, polystyrene, polyurethane, polyvinyl alcohol, polyacrylonitrile, polyamide, polymethylmethacrylate, polyethylene oxide, polytetrafluoroethylene, or copolymers thereof.
  • biocompatible polymers include, but are not limited to, polyvinylchloride, polyvinylidene fluoride, polyurethane isocyanate, polyalginate, cellulose acetate, cellulose diacetate, cellulose triacetate, cellulose nitrate, polysulfone, polystyrene, polyurethane, polyvinyl alcohol, polyacrylonitrile, polyamide, poly
  • the disks provide a suitable material onto which the cells can be disposed.
  • the disks have a trabecular structure that allows the cells to grow into the medium.
  • the cells may be disposed on the surface of the membrane.
  • the disk material may consist of a variety of different types of compounds, including ceramics, carbon, and metallic substances.
  • the disks are made from carbon material coated with niobium (Nb).
  • the disk material may be a porous, metal-coated reticulated open cell foam of carbon-containing material, the metal coating selected from, but not limited to, tantalum, titanium, platinum (including other metals of the platinum group), Nb, hafnium, tungsten, and combinations thereof.
  • the coatings (which may be non-metal as well) also may include, but are not limited to, silica, nickel, alumina, gold, collagen, non-siliceous materials, and nanofibrous scaffolds, complexes, composites, or constructs formed by any variety of methods including, but not limited to, nano-magnetic deposition, nanocasting, fiberbonding, electrospinning, or any variation on these techniques.
  • the coatings can increase integrity and strength of the disks.
  • other shapes of substrates on and/or in which cells are disposed may be used, such as rectangular or square substrates.
  • the substrates can be made from various polymers, which may have a trabeculated structure. The polymers can promote or increase cell expansion, attachment, and/or viability.
  • Cell therapy presents itself as a new approach to the treatment of acute and chronic diseases. This therapeutic approach has its origins in the growing appreciation that most disease processes are not due to the lack of a single protein but develop due to alterations in complex interactions of a variety of cell products.
  • Cell therapy depends on cell and tissue culture methodologies to expand specific cells to replace important differentiated processes deranged or lost in various disease states. Recent approaches have made progress by placing cells into hollow fiber bioreactors or encapsulating membranes as a means to deliver cell activities to a patient, requiring complex extracorporeal pump systems and large bioreactor devices.
  • SIRS systemic inflammatory response syndrome
  • SIRS single system organ failure syndrome
  • This initial cell therapy device is large (12 x 4-in cylinder) and requires an additional extracorporeal pump circuit to deliver blood and plasma ultrafiltrate to the cell- containing device. These elements were designed as an add-on to current dialysis treatment in patients with ARF.
  • the successful creation of the miniature device proposed has the potential to lead to a variety of other cell therapy devices including "wearable artificial organs”.
  • Current cell therapy approaches target the use of stem cells for neurodegenerative disorders (Parkinson's, Alzheimer's), spinal cord injury, heart disease (congestive heart failure, myocardial infarct), pancreas disease (diabetes), liver disease (cirrhosis, hepatitis), kidney disease (end-stage renal disease [ESRD], ARF), blood (sickle cell anemia), muscle disorders (muscular dystrophy), skin (burns), and bones (arthritis, osteoporosis).
  • any of the embodiments of devices for use in extracorporeal circuits according to the invention can be used to treat the disease states described herein (such as the cell therapy targets described above).
  • any type of cell can be used with any of the embodiments of devices for use in extracorporeal circuits according to the invention, including, for example, a eukaryotic cell, such as a mammalian cell, such as a renal cell (e.g., a renal tubule cell).
  • the cells can treat bodily fluids in order to prevent or alleviate the disease conditions described herein.
  • FIG. 13 depicts another embodiment of an extracorporeal treatment device where the device 90 includes a housing 84 defining an interior space. Gaskets 103 interface with the housing 84 to seal the housing. There are four gaskets shown in this embodiment, but devices may have fewer or more than shown.
  • the housing 84 includes an inlet 86 for receiving a fluid and an outlet 88 for releasing a processed fluid.
  • viable cells are disposed on a substrate, in this case on disks 98.
  • the cells can be any that are described herein.
  • the disks 98 are constructed from carbon material coated with Nb, have a trabeculated shape and are porous.
  • the disks 98 also are coated with a biomatrix material, such as, for example, collagen IV.
  • the disks either can be initially separate from the housing 84 and inserted therein or can be shipped to a customer with the disks in place.
  • Scaffolds 96 retain the disks 98 within the interior space of the housing 84, and are shaped like a tuning fork in this embodiment. There are five scaffolds shown in this embodiment, but devices may have fewer or more of the them than shown. Additionally, each scaffold may hold fewer or more disks.
  • a flow separator 102 within the interior of the housing 84 is disposed between the inlet 86 and the disks 98.
  • FIG. 14 illustrates schematically another view of Figure 13.
  • the gaskets 103 have a substantially rectangular, annular shape with a rectangular aperture therethrough, and are placed about the interior space of the housing. Additionally, the gaskets can have other shapes, including the apertures formed therethrough.
  • the embodiment illustrated in Figures 13 and 14 distributes a fluid through the disks and adjacent the viable cells.
  • the flow separator 102 is designed to prevent the fluid from solely flowing through the center of the housing. Rather, as shown in the embodiment in Figure 13, the flow separator 102 has four apertures that distribute the fluid to the front, back, and sides of the housing 84. There are four apertures shown in this embodiment, but flow separators may have fewer or more apertures of various shapes. As the fluid passes through the flow separator 102, the fluid next encounters a baffle 92.
  • each baffle 92 has apertures that correspond to the number and shape and size of the disks 98; here, each baffle 92 has four circular apertures.
  • the apertures of the baffles 92 are designed to allow the fluid to pass through the disks 98 and adjacent the viable cells. Additionally, baffles may have fewer or more apertures of various shapes.
  • Fluids that may pass through this device include ultrafiltrate (derived from blood and/or peritoneal fluid) and peritoneal fluids (including peritoneal dialysate).
  • the materials of the embodiment illustrated in Figures 13 and 14 are biocompatible.
  • the materials should not leach anything upon contact with a liquid, and toxins should not make contact with biological fluid.
  • the materials can be sterilized with the use of, but not limited to, ethylene oxide, an autoclave, gamma radiation, cold sterilant, or any combination thereof.
  • Useful materials also should not crack or substantially change when transitioning from various temperatures, such as a change to room temperature.
  • the materials may include both virgin materials and materials with biocompatible coatings.
  • the housing may be produced from a biocompatible material such as polycarbonate, polysulfone, or acrylic.
  • the baffles and flow separator may be produced from a material which includes, but is not limited to, polycarbonate, polysulfone, or acrylic.
  • the scaffolds may be produced from a material such as polypropylene.
  • the gaskets may be produced from a material such as ethylene propylene diene monomer, tetrafluoroethylene perfluoromethyl vinyl ether, polysteel, or silicone rubber.
  • the substrates on which the cells are located (in this case, the disks) can be made from a variety of materials, including ceramics, carbon, and metallic substances.
  • the substrate may be made from a porous, metal-coated reticulated open cell foam of carbon-containing material, the metal coating selected from, but not limited to, tantalum, titanium, platinum (including other metals of the platinum group), Nb, hafnium, tungsten, and combinations thereof.
  • the coatings (which may be non-metal as well) also may include, but are not limited to, silica, nickel, alumina, gold, collagen, non-siliceous materials, and nanofibrous scaffolds, complexes, composites, or constructs formed by any variety of methods including, but not limited to, nano-magnetic deposition, nanocasting, fiberbonding, electro spinning, or any variation on these techniques.
  • substrates on and/or in which cells are disposed may be used, such as rectangular or square substrates.
  • the substrates can be made from various polymers, which may have a trabeculated structure. The polymers can promote or increase cell expansion, attachment, and/or viability.
  • the components of the embodiment in Figures 13 and 14 may be produced from a biocompatible material which includes, but is not limited to, polycarbonate, polystyrene, high density polyethylene, low density polyethylene (such as any polyethylene composite), polysulfone, polypropylene, stainless steel, aluminum, titanium alloy, acrylic, syndiotactic polystyrene (such as any polystyrene composite), high impact polystyrene, ultra high molecular weight polyethylene, polyamides, polybutylene terephthalate, polyester alloy, polyetherimide, polyetheretherketone, polyphenlene ether, polyether sulfone, polyphenylsulfone, thermoplastic polyurethane, polyvinyl chloride, polytetrafluoroethylene, perfluoro alkoxy, silicone rubber, ethylene propylene diene monomer, polyvinyl fluoride, ethylene-tetrafluoroethylene, fluorinated ethylene propylene, ethylene-chlorotri
  • the substrate in this embodiment can be shipped to the site of use with cells grown on the substrate and in a cryopreserved state.
  • the substrate, and the cells thereon can be thawed and loaded into the device.
  • This configuration facilitates ease of manufacturing, shipping, and storage, as the pre-grown cells on the substrate can be shipped and stored in a frozen state.
  • Figure 15 illustrates schematically another embodiment where the entire device 190 can be freezable or cryopreserved.
  • the device 190 includes a housing 184 defining an interior space, a lid 120 to cover the housing 184, and a gasket 203 to seal together the lid 120 and the housing 184.
  • the gasket 203 has a substantially rectangular, annular shape with a rectangular aperture therethrough, and is placed about the interior space of the housing. Additionally, the gasket can have other shapes, including the apertures formed therethrough. Alternatively, two gaskets may be used to maintain a seal.
  • FIG 16 illustrates an exploded view of the embodiment shown in Figure 15.
  • the housing 184, lid 120, and gasket 203 are held together by nuts 124 and bolts 126, which are placed in grooves of the housing 184, lid 120, and gasket 203.
  • each bolt can be approximately 1 inch without interior threading.
  • the bolts can be longer or shorter or with interior threading.
  • the housing 184 includes an inlet 186 for receiving a fluid and an outlet 188 for releasing a processed fluid.
  • viable cells are disposed on a substrate, in this case on disks (not shown).
  • the disks are constructed from carbon material coated with Nb, have a trabeculated shape and are porous.
  • Each disk can have a diameter of 8mm, although the size of the disk can be greater or lesser in size.
  • the disks also are coated with a biomatrix material, such as, for example, collagen IV.
  • the disks may be constructed from ceramics or metallic substances, or the disk may be a porous, metal-coated reticulated open cell foam of carbon-containing material, the metal coating selected from tantalum, titanium, platinum (including other materials of the platinum group), Nb, hafnium, tungsten, and combinations thereof.
  • other shapes of substrates on and/or in which cells are disposed may be used, such as rectangular or square substrates.
  • the substrates can be made from various polymers, which may have a trabeculated structure.
  • the polymers can promote or increase cell expansion, attachment, and/or viability.
  • This embodiment is typically shipped to a customer in a frozen state with the disks in place. Again, this provides a benefit in manufacturing, shipping, and storage such that the completed device, with pre-grown cells in place, can be shipped in a frozen state to the site of use. A medical professional then thaws the entire device prior to use.
  • Scaffolds 196 retain the disks within the interior space of the housing 184, and are shaped like a tuning fork in this embodiment. There are five scaffolds shown in this embodiment, but devices may have fewer or more of the them than shown. Additionally, each scaffold may hold any number of disks.
  • a flow separator 202 within the interior of the housing 184 is disposed between the inlet 186 and the disks. When fluid enters the housing 184 through the inlet 186, the flow separator 202 provides desirable flow dynamics to allow the fluid to pass through the disks and adjacent the viable cells. Additionally, baffles 192 are disposed between the inlet 186 and the disks to further direct the fluids through the disks.
  • Each of the baffles 192 is adjacent or proximate to a scaffold 196.
  • the housing 184 also includes ports 194.
  • the ports 194 allow for pressure release, fluid expansion, extracorporeal flow, and analysis probes. Additionally, the device may have fewer, more, or no ports.
  • the inlet 186, outlet 188, and ports 194 are luer fittings. These components can be selectively attached or detached from tubing or grooves. Alternatively, the inlet 186, outlet 188, and ports 194 need not be luer fittings. In certain situations, an adhesive (not shown) can be used with luer fittings, such as the inlet 186, outlet 188, and ports 194 in this figure, to further maintain a seal.
  • each baffle 192 has four circular apertures that correspond to the number and shape and size of the disks.
  • baffles 192 are designed to allow the fluid to pass through the disks and adjacent the viable cells. Additionally, baffles may have fewer or more apertures of various shapes. After the fluid passes through each of the baffles 192 and disks throughout the housing 184, the processed fluid exits the housing 184 through the outlet 188. Fluids that may pass through this device include ultrafiltrate (derived from blood and/or peritoneal fluid) and peritoneal fluids (including peritoneal dialysate).
  • the materials of the embodiment illustrated in Figures 15 and 16 are biocompatible.
  • the materials should not leach anything upon contact with a liquid, and toxins should not make contact with biological fluid.
  • the materials can be sterilized with the use of, but not limited to, ethylene oxide, an autoclave, gamma radiation, cold sterilant, or any combination thereof.
  • Useful materials also should not crack or substantially change when transitioning from various temperatures, such as a change to room temperature.
  • the materials may include both virgin materials and materials with biocompatible coatings.
  • the housing may be produced from a biocompatible material such as polycarbonate, polysulfone, or acrylic.
  • the baffles and flow separator may be produced from a material which includes, but is not limited to, polycarbonate, polysulfone, or acrylic.
  • the gasket or gaskets may be produced from a material which includes, but is not limited to, ethylene propylene diene monomer, tetrafluoroethylene perfluoromethyl vinyl ether, polysteel, or silicone rubber.
  • the substrates on which the cells are located (in this case, the disks) can be made from a variety of materials, including ceramics, carbon, and metallic substances.
  • the substrate may be made from a porous, metal-coated reticulated open cell foam of carbon-containing material, the metal coating selected from, but not limited to, tantalum, titanium, platinum (including other metals of the platinum group), Nb, hafnium, tungsten, and combinations thereof.
  • the coatings (which may be non-metal as well) also may include, but are not limited to, silica, nickel, alumina, gold, collagen, non-siliceous materials, and nanofibrous scaffolds, complexes, composites, or constructs formed by any variety of methods including, but not limited to, nano-magnetic deposition, nanocasting, fiberbonding, electro spinning, or any variation on these techniques.
  • the bolts may also be constructed from a material which includes, but is not limited to, stainless steel.
  • the scaffolds may be produced from a material such as polypropylene.
  • the inlet, outlet, and ports may be produced from a material which includes, but is not limited to, polycarbonate or polypropylene.
  • the adhesive may be produced from a material which includes, but is not limited to, polyurethane adhesive or silicone adhesive.
  • the components of the embodiment in Figures 15 and 16 may be produced from a biocompatible material which includes, but is not limited to, polycarbonate, polystyrene, high density polyethylene, low density polyethylene (such as any polyethylene composite), polysulfone, polypropylene, stainless steel, aluminum, titanium alloy, acrylic, syndiotactic polystyrene (such as any polystyrene composite), high impact polystyrene, ultra high molecular weight polyethylene, polyamides, polybutylene terephthalate, polyester alloy, polyetherimide, polyetheretherketone, polyphenlene ether, polyether sulfone, polyphenylsulfone, thermoplastic polyurethane, polyvinyl chloride, polytetrafluoroethylene, perfluoro alkoxy, silicone rubber, ethylene propylene diene monomer, polyvinyl fluoride, ethylene-tetrafluoroethylene, fluorinated ethylene propylene, ethylene-chlorotri
  • the embodiment of the present invention illustrated in Figures 15 and 16 accomplishes freezing or cryopreservation of the entire device 190 in order to maintain cell viability.
  • the device 190 can be viable in a liquid nitrogen vapor phase at a temperature range of negative 90 to negative 150 degrees Celsius; alternatively, the device 190 can be stored in the liquid phase at negative 190 degrees Celsius.
  • the device 190 is also biocompatible, maintains seal without leaking or cracking, and withstands sterilization and pressure.
  • the device 190 may be stored in a sterile cryo-compatible bag for freezing and thawing. Additionally or alternatively, the device 190 may incorporate (temporarily or permanently) a one way outward valve at the inlet.
  • the valve allows for outlet venting during the freeze/thaw process while keeping the outside, non-sterile air from entering the device.
  • the embodiments of the present invention illustrated in Figures 13-16 may also include a pre- and/or a post-filtration unit integrated with the housing, thus creating a porous barrier between the viable cells and the body fluid flowing into the treatment device.
  • the filtration unit prevents the cells within the housing from provoking an immunological response and produces an ultrafiltrate that provides nutrients and oxygen to the cells.
  • the flow separator can either be separate from or integrated with the housing.
  • the substrate on which the cells are disposed can be any shape, such as rectangular or square.
  • Devices of the present invention as illustrated in Figures 13-16 may be sealed with a snap-fit or screw-fit design rather than using gaskets, nuts, and/or bolts.
  • the snap-fit or screw-fit design can incorporate a holding system for the substrates (e.g., disks) into the housing, such as a combined scaffold/baffle assembly. Further, the design can have a flow separator integrated with the housing.
  • the embodiments of the present invention illustrated in Figures 13-16 can employ the use of dynamic culture and seeding methods, such as, for example, for renal cell therapy.
  • the disks of the embodiments of Figures 13-16 can be cultured under rotational flow.
  • Figures 17 A and B depict another embodiment 290 of the present invention where viable cells are seeded in hollow fibers 1152 contained within a housing 284.
  • the housing 284 includes an inlet 286 for receiving a fluid, an outlet 288 for releasing a processed fluid, and two ports 294, 296.
  • Hollow fibers 1152 connect the inlet 286 and the outlet 288.
  • the hollow fibers 1152 define a luminal space referred to as an intracapillary space (ICS) 1140.
  • ICS intracapillary space
  • the space surrounding the hollow fibers 1152 is the extracapillary space (ECS) 1142 and is within the housing 284.
  • ECS extracapillary space
  • an adhesive (not shown) can be used to occupy the spaces between the hollow fibers 1152 at the inlet 286 and at the outlet 288 of the device 290.
  • the adhesive holds the hollow fibers 1152 in place and defines the ICS and the ECS of the device 290.
  • the device 290 can sustain cells 300 for incorporation into ex vivo circuits or implantation.
  • Viable cells 300 are seeded within the ICS 1140 of at least one hollow fiber 1152.
  • the cells 300 can be adherent cells.
  • the cells 300 are grown on particles 298, such as beads or other microcarriers, and are seeded into the ICS 1140 while on the particles 298.
  • microcarriers include, but are not limited to, HyQ® SpheresTM microcarriers (available from HyClone, Inc., South Logan, Utah and SoloHill Engineering, Inc., Ann Arbor, MI), Cultispher® microcarriers (available from HyClone, Inc. South Logan, Utah), Cytodex® microcarrier beads (available from GE Healthcare Bio-Sciences Corp., Piscataway, NJ), and Glass microcarrier beads (available from Sigma-Aldrich, Inc., St. Louis, MO).
  • the HyQ® SpheresTM microcarrier is a cross-linked, polystyrene microcarrier. It can be optionally coated with collagen or animal-derived component free materials. Further, it can have a cationic charge layering surface modification.
  • the Cultispher® microcarriers have larger surface areas for attachment and are dissolvable for cell retention. They come in three types (G: basic gelatin cross linked, S: large pore cross linked, and GL: high temperature cross-linked).
  • the Cytodex® microcarrier bead is a dextran, cross-linked microcarrier. It has positively charged Diethylaminoethyl (DEAE) groups throughout the matrix.
  • the Glass microcarrier beads can be of varying size and density.
  • the cells 300 are within the device 290, they are located within a biomatrix material 292, which itself is disposed within the ICS 1140. Thus, the cells are suspended in the biomatrix material 292 or physiologic buffer (e.g., a 3-D matrix suspension) in the hollow fibers 1152.
  • a biomatrix material 292 or physiologic buffer include, but are not limited to, alginate solution, gelled alginate solution, nutragen, matrigel, collagen I or any three-dimensional forming or formed biocompatible matrix.
  • the hollow fibers 1152 of this or other hollow-fiber embodiments of the invention can have any of the following design characteristics.
  • the hollow fibers can have immunoprotective properties, and cells can be cultured on the inside of the fibers.
  • the pores through the hollow fibers can be of a size within the range of about 0.01 ⁇ m to about 0.1 ⁇ m. Pore sizes also can be measured as a molecular weight cutoff (MWCO) and can be of a type useful for ultrafiltration. MWCO and size are loosely related with 0.01 ⁇ m equating to a MWCO of about 70,000 kD and 0.1 ⁇ m equating to a MWCO of about 500,000 kD.
  • MWCO molecular weight cutoff
  • the hollow fibers typically have a MWCO of about 40,000-50,000 kD.
  • the hollow fibers can function as a barrier between the nutrient supply source and the cells.
  • the biomatrix material and/or cells can be inserted into the device at the point of manufacture or, alternatively, at the clinical site. If the cells are inserted at the point of manufacture, the device can be frozen and delivered in such a state. Then, the entire device is thawed prior to use by the medical professional. If the cells are inserted at the clinical site, the cells can be cryopreserved separately from the device.
  • cryopreserved cells are thawed at the clinical site, when they are to be used, mixed with the biomatrix material and injected (e.g., with a syringe) into the device.
  • the cryopreserved cells can be pre-mixed with the biomatrix material and both components can be thawed when used.
  • cryopreserved cells, grown on microcarriers are thawed and, if necessary, mixed with a three-dimensional biomatrix, such as alginate. This mixture is injected into the device, and the mixture is allowed to gel.
  • the materials of the embodiment illustrated in Figures 17 A and B are biocompatible.
  • the materials should not leach anything upon contact with a liquid, and toxins should not make contact with biological fluid.
  • the materials can be sterilized with the use of, but not limited to, ethylene oxide, an autoclave, gamma radiation, cold sterilant, or any combination thereof.
  • Useful materials also should not crack or substantially change when transitioning from various temperatures, such as a change to room temperature.
  • the materials may include both virgin materials and materials with biocompatible coatings.
  • the housing or cartridge may be produced from a biocompatible material such as polycarbonate.
  • the hollow fibers e.g., semi-permeable encapsulating membranes
  • the hollow fibers may be produced from a material which includes, but is not limited to, polysulfone, poloyetherimide, polyether sulfone, helixone, polyalginate, polyvinylchloride, polyvinylidene fluoride, polyurethane isocyanate, cellulose acetate, cellulose diacetate, cellulose triacetate, cellulose nitrate, polysulfone, polystyrene, polyurethane, polyvinyl alcohol, polyacrylonitrile, polyamide, polymethylmethacrylate, polytetafluoroethylene, polyethylene oxide, or copolymers thereof.
  • the inlet, outlet, and ports may be produced from a material which includes, but is not limited to, polycarbonate or polypropylene.
  • the adhesive may be produced from a material which includes, but is not limited to, polyurethane adhesive or silicone adhesive.
  • the components of the embodiment in Figures 17A and B, with the exception of the viable cells may be produced from a biocompatible material which includes, but is not limited to, polycarbonate, polystyrene, high density polyethylene, low density polyethylene (such as any polyethylene composite), polysulfone, polypropylene, stainless steel, aluminum, titanium alloy, acrylic, syndiotactic polystyrene (such as any polystyrene composite), high impact polystyrene, ultra high molecular weight polyethylene, polyamides, polybutylene terephthalate, polyester alloy, polyetherimide, polyetheretherketone, polyphenlene ether, polyether sulfone, polyphenylsulfone, thermoplastic polyurethane, polyvin
  • the extracorporeal devices described herein can be used with extracorporeal circuits as illustrated in Figures 18A and B ( Figures 18A and B show the device of Figure 13, but the circuits can be used with other devices according to the invention, such as, for example, the devices of Figures 15 and 16).
  • the blood from a subject e.g., an animal or a human
  • the blood is then circulated through a first hemofilter 106 having hollow fibers therein defining an ICS and an ECS.
  • the blood passes through the ICS, and is split into a filtered blood fraction and an ultrafiltrate fraction.
  • the flow rate of ultrafiltrate generated by the first hemofilter 106 is controlled by a second pump 108 that passes the ultrafiltrate from the first hemofilter, through a thermal regulator 110 for maintenance of temperature within the device 90 illustrated in Figure 13, into the device 90 where the ultrafiltrate flows across the viable cells on the disks 98, and into a second hemofilter 112.
  • the second hemofilter 112 outlet flow rate is controlled by a third pump 114 to allow for maximal ultrafiltrate filtration to pass through to the ECS of the second hemofilter 112, while maintaining physiologic pressures.
  • the processed ultrafiltrate upon exiting the second hemofilter 112, the processed ultrafiltrate is combined with the filtered blood, which came directly from the first hemofilter 106, and is returned to the subject.
  • the filtered blood from the first hemofilter 106 flows directly to the second hemofilter 112 and is combined with the processed ultrafiltrate.
  • the combined processed ultrafiltrate and filtered blood exit the second hemofilter 112 and are returned to the subject.
  • the circuits of Figures 18A and B utilize, for example, central line catheter access (due to the blood flow rates desired to generate beneficial ultrafiltrate flow rates to sustain the nutritional and oxygen needs of the cells) and a multiple pump system with pre- and post- device hemofilters.
  • the actual treatment device portion of the circuit utilizes ultrafiltrate, rather than blood, thus eliminating potential clotting in the cell unit. It also isolates the cells in the device from immunological insult.
  • the therapy circuit can be readily managed in an intensive care unit (ICU) setting.
  • ICU intensive care unit
  • the premise of this cell-based therapy is that as the ultrafiltrate passes through the cell chamber of the treatment device, systemic small-molecular-weight molecules in the ultrafiltrate, which have been produced due to a disease state, such as a septic disease state, trigger a therapeutic response in the cells within the treatment device, which in turn secrete small-molecular-weight molecules into the passing ultrafiltrate.
  • This processed ultrafiltrate is then refiltered across the hollow fiber membrane of the second hemofilter and is then joined to the filtered blood and returned to the systemic circulation, with the released cell factors providing therapeutic value, for example, to the septic state.
  • the lines may circulate other body fluids, for example, peritoneal fluid that is removed from the peritoneum. Peritoneal fluid is circulated outside the body, where the cells within the treatment device are able to deliver the desired molecules to the fluid. This processed fluid is then reintroduced into the peritoneum.
  • the extracorporeal devices described herein can be used with the extracorporeal circuits of Figures 19-21 and Figure 55.
  • Figures 19 and 20 show the device of Figure 13, but the circuits can be used with other devices according to the invention, such as, for example, the device of Figures 15 and 16.
  • the circuit of Figure 21 shows the device of Figure 13, but it too can be used with other devices according to the invention, such as, for example, the device of Figures 15 and 16.
  • the circuit of Figure 55 shows the device of Figures 15 and 16, but it can be used with other devices according to the invention, such as, for example, the device of Figure 13.
  • the extracorporeal circuits add peritoneal dialysis fluid to peritoneal fluid.
  • peritoneal dialysis solutions can be used, including, but not limited to, peritoneal dialysate fluid with 1.5% dextrose solution (Deflex®; code 054-20201, Na
  • peritoneal dialysate fluid with 2.5% dextrose solution (Deflex®; code 054-20202, Na 132mEq/L, Ca 3.5mEq/L, Mg 0.5mEq/L, Cl 95mEq/L, Lactate 40mEq/L, Dextrose 2.5%, available from Fresenius Medical Care North America, Waltham, MA) can be used.
  • the subject's belly is filled with about IL to about 3L of peritoneal dialysis fluid, which is maintained by an additional pump. It is infused at a rate to generally match the reabsorption by the subject. This allows for a volume to be maintained in the belly for recirculation.
  • peritoneal fluid and peritoneal fluid ultrafiltrate is used in the description of Figures 19-21 and Figure 55, it should be understood that the circuits can be used with or without the addition of peritoneal dialysis fluid such that peritoneal fluid ultrafiltrate with or without addition of peritoneal dialysis fluid (either before or after ultrafiltration) and/or peritoneal fluid with or without addition of peritoneal dialysis fluid can be used.
  • the peritoneal fluid of a subject is pumped via a first pump 134 through a first dialysis filter 136 having hollow fibers therein defining an ICS and an ECS.
  • the pump can pump at a rate including, but not limited to, of about 25mL/min to about 200mL/min.
  • the peritoneal fluid passes through the ICS of the dialysis filter 136, and is split into an unfiltered peritoneal fluid fraction and an ultrafiltrate peritoneal fluid fraction.
  • the flow rate of peritoneal fluid ultrafiltrate generated by the first dialysis filter 136 is controlled by a second pump 138 that passes the peritoneal fluid ultrafiltrate from the first dialysis filter 136, through an optional thermal regulator 140 for maintenance of temperature within the device 90 illustrated in Figure 13, and into the treatment device 90.
  • the peritoneal fluid ultrafiltrate flows across the viable cells on the disks 98, exits the device, and enters a second dialysis filter 142.
  • the thermal regulator 140 need not be used.
  • the processed peritoneal fluid ultrafiltrate Upon exiting the second dialysis filter 142, the processed peritoneal fluid ultrafiltrate has been combined with the unfiltered peritoneal fluid, which came directly from the first dialysis filter 136, and is returned to the subject.
  • Peritoneal dialysis fluid is infused via a pump (not shown) at a point before the first pump 134.
  • the extracorporeal circuit of Figure 21 is similar to the circuit of Figure 19, except the second pump 138 is placed downstream from the treatment device 90 rather than upstream. [0151]
  • the circuit of Figure 20 also is similar to that shown in Figure 19.
  • the peritoneal fluid of a subject is pumped via a first pump 134 through a first dialysis filter 136 having hollow fibers therein defining an ICS and an ECS.
  • the first pump 134 can pump at a rate including, but not limited to, about 5mL/min to about 200mL/min.
  • the peritoneal fluid is forced from the ICS, across the hollow fibers, to the ECS of the first dialysis filter 136, and all fluid is filtered. Only a portion of the filtered peritoneal fluid, however, moves on to the treatment device 90.
  • the flow rate of peritoneal fluid ultrafiltrate generated by the first dialysis filter 136 is controlled by a second pump 138 that passes some of the peritoneal fluid ultrafiltrate from the first dialysis filter 136, through an optional thermal regulator 140 for maintenance of temperature within the device 90 illustrated in Figure 13, and into the treatment device 90.
  • the peritoneal fluid ultrafiltrate flows across the viable cells on the disks, exits the device 90, and enters a second dialysis filter 142 with hollow fibers defining an ICS and an ECS.
  • the thermal regulator 140 need not be used.
  • the excess ultrafiltrate that was not delivered to the cells in the treatment device 90 is also passed on to the second dialysis filter 142 and mixed with the processed ultrafiltrate from the treatment device 90.
  • the remixed ultrafiltrate is filtered a second time by being forced across the hollow fibers from the ECS into the ICS of the second dialysis filter 142.
  • part of the fluid is returned to the subject under the control of another pump 234.
  • the remaining fluid is recirculated to the beginning of the circuit.
  • This circuit may decrease the stress on the abdominal cavity of the subject while still allowing for the flow rates desirable to maintain the circuit and cells.
  • the circuit also allows for ultrafiltration to further protect the subject from bacteria and fibrin produced in the belly of the subject.
  • the pump 304 passes the blood through a thermal regulator 310 for maintenance of temperature within the treatment device 290, illustrated in Figures 17 A and B, which has hollow fibers 1152 therein defining an ICS 1140 and an ECS 1142.
  • the blood enters the ECS 1142 of the treatment device 290 through a first port 294, passes by the outside of the hollow fibers 1152, and then exits the device 290 at a second port 290. This processed blood is then returned to the subject.
  • Systemic small-molecular-weight molecules from the blood that have been produced due to the disease state of the subject trigger a therapeutic response of the viable cells, which in turn secrete small-molecular- weight molecules that cross the hollow fiber membrane 1152 and are returned to the system circulation of the subject, providing therapeutic value to the disease state.
  • the circuit of Figure 22 utilizes, for example, a peripheral catheter for blood access (due to the desirable blood flow rates of less than about 50 mL/min to support the treatment device 290). Peripheral access for this therapy circuit allows it to be managed in a non-ICU-based hospital setting.
  • the lines of the circuit shown in Figure 22 may circulate other body fluids, for example, peritoneal fluid that is removed from the peritoneum. Peritoneal fluid is circulated outside the body, where the cells within the treatment device is able to deliver the desired molecule(s) to the fluid. This processed fluid is then reintroduced into the peritoneum.
  • An alternative extracorporeal circuit is shown in Figure 55. In this circuit design, the peritoneal fluid of the subject (e.g., animal or human) is pumped via a first pump 134 through a dialysis filter 136 having hollow fibers therein defining an ICS and an ECS.
  • the first pump 134 can pump at a rate including, but not limited to, about 5 mL/min to about 200 mL/min.
  • the peritoneal fluid to sustain the treatment device 190 is pulled across the hollow fiber membrane from the ICS to the ECS by a second pump 138 and moves through an optional warming device 140 before being delivered to the treatment device 190.
  • the flow rate of peritoneal fluid ultrafiltrate generated by the first dialysis filter 136 is controlled by the second pump 138.
  • the peritoneal fluid ultrafiltrate flows across the viable cells on the disks and exits the device 190.
  • the processed ultrafiltrate continues passage via another pump 234 that controls delivery to the patient.
  • FIG. 1 Fabrication and in vitro testing of a second prototype with cryopreservation storage capacity.
  • Prototype development of a second cell therapy device is also schematized in Figure 1.
  • This second device illustrated in Figures 2 and 3, contains a series of disks forming a trabecular structure on which cells attach and grow.
  • the use of individual disks allows for the capability to deliver a range of therapeutic doses taking into account different ages, sizes, and degrees of disease states of the subject.
  • Preliminary data suggest up to 1.0 x 10 7 cells can grow on each disk, which is made of a new biomaterial carbon coated with Nb.
  • Ten disks can be placed within a cartridge that acts as a semipermeable membrane to produce plasma ultrafiltrate.
  • the blood from a patient can be through an Aterio Venous (AV) line that could be used in dialysis.
  • AV Aterio Venous
  • VV venous to veneous
  • an artery 70 and vein 72 can be used to extract blood and reintroduce blood for an extracorporeal circuit 74.
  • Various suitable connections may be used to increase the diameter of the conduit.
  • the device 30 is illustrated as being attached to the largest conduit 76.
  • the lines may circulate body fluids, for example, peritoneal fluid that is removed from the peritoneum and circulated outside the body where the device is able deliver the desired molecule to the fluid and then reintroduced into the peritoneum.
  • Cells would be grown on Nb-coated carbon based, disk-shaped scaffolds. These cell- seeded disks would be grown in culture and then cryopreserved. Disks could be shipped frozen to clinical sites were they could be kept frozen until required for cell- based therapy. Thawed disks could be placed in the device housing just prior to therapy. The number of disks used per therapy session (loaded into the holding device) could be varied depending on the cellular dose required. 2. Cells resuspended in a biomatrix 3-D gel would be seeded into the lumen of a hollow fiber bioreactor.
  • MDCK immortalized renal cell line
  • disks Prior to cell seeding, disks were coated with either collagen I, collagen IV, laminin, or matrigel. Seeding densities were 10 6 cells/disk. The disks were maintained in a spinner culture for 1 week. Cell- seeded disks were then cryopreserved, stored in liquid nitrogen and then thawed, with a non-stir rest period under tissue culture conditions of 2-4 hours post thaw. Disks were then reinstated in spinner culture bottles and assayed for cell density 24 hours post thaw. Lactate production was utilized to determine cell expansion on the disks. MDCK cell densities on disks with various bio-matrix coatings pre and post cryopreservation are depicted in the graph of Figure 9.
  • PPTC primary porcine renal proximal tubule cells
  • Hollow fiber bioreactor containing cells resuspended in a biomatrix 3-D gel [0165] Cells from an immortalized renal cell line (MDCK) resuspended in either 1) nutragen (collagen I (3.12 mg/mL) gel supplemented with laminin (100 ⁇ g/mL) and collagen IV (100 ⁇ g/mL)) or 2) matrigel were seeded into hollow fiber bioreactors at a density of 2.06 x 10 7 cells/mL of gel. Cell loaded bioreactors were maintained in culture for 8 (matrigel) and 13 (nutragen) days, with cell density assessed via lactate production. Results are depicted in the Figure 12. BREC refers to Bioartificial Renal Epithelial Cell.
  • Example 2 Testing of Cell-Seeded Nb-Coated, Carbon-Based, Disk-Shaped Substrates
  • BRECS-d the design of the device illustrated in Figure 13 (referred to in this Example as the "BRECS-d” or "BRECS"), which is constructed such that the disks can be cryopreserved, allowing for a simplified manufacturing process and ease of clinical storage and deployment.
  • the BRECS-d therapy circuit typically utilizes central line catheter access (due to the blood flow rates desired to generate beneficial ultrafiltrate flow rates to sustain the nutritional and oxygen needs of the cells)) and a multiple pump system with pre- and post-BRECS hemofilters, the actual BRECS-d portion of the circuit utilizes ultrafiltrate rather than blood, thus eliminating potential clotting in the cell unit as well as further isolating the cells in the unit from immunological attack.
  • BRECS-d blood therapy circuits illustrated, for example, in Figures
  • Tissue engineering and cell therapy is a new and exciting approach to the treatment of acute and chronic diseases.
  • the potential success of this therapeutic approach lies in the growing appreciation that most disease processes are not due to the lack of a single protein but develop due to alterations in complex interactions of a variety of cell products.
  • Cell therapy is dependent on cell and tissue culture methodologies to expand specific cells to replace important differentiated processes deranged or lost in various disease states.
  • Recent approaches have made progress by placing cells into hollow fiber bioreactors or encapsulating membranes as a means to deliver cell activities to a patient.
  • a reasonable extension of this approach is to add cell therapy to the current successful renal substitution processes of hemodialysis and hemofiltration.
  • the research outlined herein is a new therapeutic approach based upon the thesis that renal tubule cells play an important immunologic regulatory role in septic shock. Accordingly, the effect of renal tubule cell therapy improves cardiovascular parameters, immunologic activity, and ultimate survival rate of normal, non-uremic large animals in septic shock.
  • the approach is based upon the observation that severe septic shock results in ATN and ARF within hours of bacteremia in a porcine model of septic shock.
  • ARF develops early in the time course of septic shock, a time frame not appreciated clinically since it takes several days to observe a rise in blood urea nitrogen (BUN) and serum creatinine after the acute insult.
  • BUN blood urea nitrogen
  • the disorder of ARF, or ATN may be especially amenable to cell therapy in conjunction with continuous hemofiltration techniques, since acute hemodialysis or hemofiltration has yet to reduce the mortality rate of ATN below 50 percent, despite advances in synthetic materials and extracorporeal circuits.
  • ATN develops predominantly due to the injury and necrosis of renal proximal tubule cells. The early replacement of the functions of these cells during the episode of ATN, which develops concurrently with septic shock, will provide almost full renal replacement therapy in conjunction with hemofiltration.
  • metabolic activity such as ammoniagenesis and glutathione reclamation
  • endocrine activity such as vitamin D 3 activation
  • cytokine homeostasis is contemplated to provide additional physiological replacement activities to change the current natural history of this disease process.
  • the kidney is derived embryologically from dorsal mesoderm, a collection of cells also important in the development of bone marrow stem cells.
  • the maturation of cells responsible for erythropoietin synthesis and activation of 1,25-(OH) 2 vitamin D 3 in the kidney is reflective of this embryonic origin.
  • the kidney is the major antibody-producing organ.
  • mammalian renal proximal tubule cells are immunologically active. They are antigen-presenting cells that possess co-stimulatory molecules and that synthesize and process a variety of inflammatory cytokines.
  • ARF, and ATN that results in the loss of the kidney's immunoregulatory function results in a propensity to develop SIRS, sepsis, MOF, and a high risk of death.
  • a rise in sepsis events can be observed from 3% to nearly 60% in patients who develop ARF during the post- op course following open heart surgery.
  • a counterpart to this loss of immunologic function has been observed in chronic renal insufficiency and end-stage renal disease, which are clearly pro-inflammatory states. The degree of inflammation in these patient populations has been highly correlated to mortality rates.
  • the loss of renal tubule cells, rather than loss of filtration and clearance function may be the cause of this inflammatory dysregulation observed in these patients as well.
  • the bioartificial kidney set-up consists of a filtration device (a conventional hemofilter) followed in series by the tubule RAD unit. Specifically, blood is pumped out of a large animal using a peristaltic pump.
  • the blood then enters the fibers of a conventional hemofilter, where ultrafiltrate is formed and delivered into the lumen of the hollow fibers within the RAD, downstream to the conventional hemofilter.
  • Processed ultrafiltrate exiting the RAD is collected and discarded as urine.
  • the filtered blood exiting the hemofilter enters the RAD through the ECS port and disperses among the fibers of the device.
  • the processed blood travels through a third pump and is delivered back to the animal. This additional pump is required to maintain appropriate hydraulic pressures within the RAD. In this regard, the pressures of the blood and ultrafiltrate just before entry into the RAD are monitored. Heparin is delivered continuously into the blood before entering the RAD to diminish clotting within the device.
  • the RAD is oriented horizontally and placed into a temperature-controlled environment.
  • the temperature of the cell compartment of the RAD must be maintained at 37 0 C throughout its operation to ensure optimal functionality of the cells. Maintenance of a physiologic temperature is a critical factor in the functionality of the RAD.
  • the unit is able to maintain viability because metabolic substrates and low-molecular- weight growth factors are delivered to the tubule cells from the ultrafiltration fluid and the blood in the ECS.
  • immunoprotection of the cells grown within the hollow fiber is achieved due to the impenetrance of immunoglobulins and immunologically competent cells through the hollow fibers. Rejection of the cells, therefore, does not occur. This arrangement thereby allows the filtrate to enter the internal compartments of the hollow fiber network, lined with confluent monolayers of renal tubule cells for regulated transport and metabolic function. Improvements to Renal Cell Therapy Devices
  • the RAD is an extracorporeal device utilizing a standard hemofiltration cartridge containing renal tubule cells grown from adult stem/progenitor cells as confluent monolayers along the inner surface of the fibers.
  • This initial cell therapy device requires large cell numbers for seeding, is labor intensive to generate and maintain and cannot be held in a frozen state for ready deployment at a clinical site.
  • the RAD is large (12 x 4-in cylinder) and requires an additional extracorporeal pump circuit to deliver blood and plasma ultrafiltrate to the cell-containing device.
  • the RAD was designed as an add-on to current dialysis treatment in patients with ARF.
  • a miniaturized cell therapy device will not require extensive extracorporeal blood pump systems.
  • a miniaturized device that could be stored at the clinical site for immediate use is desirable to succeed as a commercial product.
  • the current RAD is stored at a central manufacturing facility at 37 0 C and must be shipped at 37 0 C to the clinical site, delaying treatment and adding to the cost of therapy.
  • Development of a cell device that can be cryopreserved and/or stored at clinical sites is a desirable feature for optimal deployment of such devices.
  • RAD therapy at the onset of bacteremia (RAD therapy is initiated at the same time that bacteria is infused) in these established animal models "prevents" the rapid deterioration from SIRS.
  • a more reproducible porcine model of septic shock in which the time to death is greater than 4-6 hours after initiation of sepsis, needs to be developed.
  • volume resuscitation protocol was increased from 100 mL/hr to 150 mL/hr immediately after the crystalloid/colloid bolus infusion at the time of bacteria administration, as detailed below.
  • tissue-specific consequences of sepsis with or without RAD therapy were evaluated with bronchoalveolar lavage (BAL) to better understand the immunoregulatory role of renal tubule cell therapy.
  • BAL specimens were used to assess pulmonary microvascular damage and inflammation in response to SIRS. Data detailed below demonstrated that renal cell therapy was associated with less protein leak from damaged blood vessels and less inflammation in BAL fluid samples, as well as improvement of other cardiovascular effects of SIRS.
  • the improved porcine model of septic shock was used to preliminarily assess the multiorgan effects of RAD intervention.
  • the following data are derived from a total of 14 animals: 7 sham controls and 7 RADs (second cartridge containing porcine renal epithelial cells). Cardiovascular Parameters
  • BAL fluid was obtained at the time of death and evaluated for protein content as a parameter of microvascular damage, various inflammatory cytokines and the absolute number of polymorphonuclear cells (PMNs).
  • PMNs polymorphonuclear cells
  • Protein TNF- ⁇ IL-6 IL-8 fe/g/mL (pg/mL) (pg/ml) (pg/mL) (pg/ml)
  • the higher levels of IL-8 may reflect the effect of sepsis in increasing IL-8 release from renal epithelial cells.
  • a more reproducible porcine model of sepsis was developed with longer survival times to compare therapeutic efficacy of new device formulations, which will be the focus of the next research steps.
  • tissue-specific inflammatory effects of therapy with BAL fluid analysis were incorporated into this model system.
  • a BRECS unit that could be stored at the clinical site for immediate use will improve its ability to be used in a clinical setting.
  • Development of a cell device that can be cryopreserved and stored at clinical sites is desirable clinically.
  • the most advanced design of these BRECS prototypes (the carbon disk-based BRECS-d) was then tested in 2 animals, under the established porcine model of sepsis, utilizing an extracorporeal system that incorporated ultrafiltrate delivery, rather than blood, to the BRECS-d unit.
  • PPRECs were seeded on Nb-coated carbon-based disks at a density of 10 6 cells/disk. Prior to cell seeding, disks were coated with collagen IV. Preliminary studies determined that collagen IV-coated disks displayed superior cell growth as compared to disks coated with collagen I, laminin, or matrigel. The disks were maintained in a spinner culture for up to 7 weeks. After the initial post-seeding expansion, cell number, as determined by lactate production, remained stable over the 7-week period ( Figure 28).
  • Figures 29 A-D show the disks to be covered uniformly, as assessed by these two staining methods.
  • Figure 29 A shows a cell- seeded carbon disk after several weeks in
  • FIG. 29B is a close-up view of a section of Figure 29A.
  • Figures 29C and D show a collagen IV-coated sham carbon disk and a cell-seeded carbon disk, respectively, after staining with crystal violet. Cells are readily visualized via the dark staining on the cell disk as compared to no staining on the sham disk.
  • PPRECs were seeded on Nb-coated carbon-based disks, pre- coated with collagen IV, at a cell density of 10 6 cells/disk. The disks were maintained in a Starwheel spinner culture for two weeks.
  • the Starwheel system was designed so that four disks were held by a tuning fork ⁇ i.e., a scaffold) in a vertical position with up to eight scaffolds per Starwheel flask. After two weeks, five scaffolds, each containing four disks, were placed in a BRECS-d unit for pre-cryopreservation oxygen and glucose consumption and lactate production rate determination. The cell-seeded disks were then cryopreserved in groups consisting of four disks per scaffold system, stored in liquid nitrogen for 48-72 hours, and then thawed for post-cryopreservation oxygen and glucose consumption rate assessment. The 4-disk groups were quick thawed at 37 0 C, and the five scaffold systems were placed in the BRECS-d unit illustrated in Figure 13.
  • the BRECS-d unit was perfused with media at increasing flow rates over a 2-hour period.
  • the post BRECS-d perfusate was collected for cell counts to determine cell attachment post-cryopreservation.
  • the number of released cells in the post BRECS-d perfusate was insignificant as determined by hemocytometer (data not shown).
  • Figures 30, 31, and 32 depict pre- and post-cryopreservation oxygen consumption rates, glucose consumption rates, and lactate production rates, respectively. Insofar as oxygen rates were not decreased, it is contemplated that freeze/thawing does not negatively impact cell viability of frozen cells.
  • Figures 37-39 show a comparison of lamb renal epithelial cell (LREC) active disk consumption rates versus LREC fixed disk consumption rates over time.
  • Figure 38 shows the difference in glucose consumption rates of LRECs after a freeze thaw process.
  • Figure 39 illustrates the difference in oxygen consumption rates of LRECs after a freeze thaw process.
  • Study 4 and Study 5 of Figures 38-39 were two different sets of LRECs grown on disks that underwent the cryopreservation process. These two studies demonstrated maintenance of cell viability when seeded on disks after thawing ⁇ i.e., post-cryopreservation).
  • the results compare the measured consumption rates of the cells pre- and post-freeze to show that the cells are viable after undergoing such a strenuous process. These results indicate that the cells can be frozen on disks and then be viable and metabolically active after being thawed.
  • the PPREC-conditioned media significantly decreased leukocyte activation, as determined by oxidative burst, under both non-LPS and LPS conditions compared to the fresh media condition.
  • the results are depicted in Figure 34 with DCHF expressed as relative fluorescent units (RFUs).
  • REUs relative fluorescent units
  • leukocyte oxidative burst assay generated preliminary data that could be used to show the potential for assessing differences in the activated state of systemic leukocytes
  • this assay can be cumbersome and dependent on the isolation methods employed to obtain the leukocytes to be used in this assay. Variability was found due to the isolation process itself activating the leukocytes.
  • An alternative method which could be used as an indicator of leukocyte activation has now been developed. The following protocol will be used for assessing the level of neutrophil, monocyte, and eosinophil activation in porcine peripheral blood. This approach measures the level of CDl Ib expression on the cell surface of each leukocyte population as an indicator of the "activation" level attained by the population.
  • the protocol calls for measuring the level of leukocyte surface expression by staining freshly isolated whole blood with a FITC-conjugated anti-porcine CDl Ib antibody. Red cells are then lysed and the leukocytes fixed using Becton-Dickenson's "Facs lyse" solution.
  • the blood was then either incubated immediately with a FITC-conjugated anti-porcine CDl Ib antibody or after a 30 minute exposure to PMA.
  • the PMA condition acts as a positive control for maximal stimulation of leukocytes.
  • the antibody/blood mixture was then fixed with a Becton-Dickenson's "Facs lyse" solution and the leukocyte population was analyzed for activated neutrophils, monocytes and eosinophils.
  • the intent of this data was to assess the assay ability to discern between various leukocyte cell populations and be a sensitive index of leukocyte activation. From the data generated, both of these criteria were achieved.
  • This assay will be used in two ways. First, the effect of cell therapy compared to sham controls on leukocyte activation will be assessed. Blood will be collected and leukocytes isolated at baseline (prior to bacteria infusion) and then again at 6 hours post-bacteria infusion. The intensity of fluorescent signal of FITC-conjugated anti-porcine CDl Ib antibody bound to the leukocyte surface will be analyzed by flow as an indicator of leukocyte activation and compared between sham- and cell-treated groups. The effect of an HREC-seeded BRECS-d device on leukocyte activation in an ovine anephric model is described in Example 7.
  • conditioned media will be collected from the BRECS-d units and incubated with normal, non- septic isolated porcine leukocytes under non-LPS and LPS exposure conditions.
  • Anti-porcine CDl Ib antibody staining of the leukocytes will be assessed and used as a biomarker index of the BRECS unit's ability to inhibit the activation of leukocytes. It is contemplated that the BRECS unit will function similar to the kidney, by secreting various cytokines/molecules to mediate the activation of the leukocytes.
  • the devices used in this study were seeded with human renal proximal tubule cells isolated from kidneys donated for cadaveric transplantation but found to be unsuitable for transplantation due to anatomic or fibrotic defects.
  • the results of this clinical trial demonstrated that the experimental treatment could be delivered safely under study protocol guidelines for up to 24 hours when used in conjunction with CVVH.
  • the device also demonstrated differentiated metabolic and endocrinologic activity. All but one treated patient with more than a 3-day follow-up showed improvement, as assessed by acute physiologic scores.
  • Plasma cytokine levels suggests that RAD therapy produces dynamic and individualized responses in patients depending on their unique pathophysiologic conditions.
  • Renal cell therapy improved the 28 -day mortality rate from 61% in the conventional hemofiltration-treated control group to 34% in the RAD-treated group. This survival impact continued through the 90- and 180-day follow-up periods (p ⁇ 0.04), with the Cox proportional hazard ratio indicating that the risk of death was 50% of that observed in the continuous venovenous hemofiltration (CVVH) therapy group ( Figure 36).
  • the BRECS-d design allows for the device to be both miniaturized and cryopreserved, thus eliminating the shortcomings of the original RAD design, such as needing to maintain the RAD in culture prior to clinical use, which demanded significant hands-in technical time.
  • the technology is a bioengineered device that utilizes renal cells in the treatment of diseases, such as SIRS and septic shock.
  • the target treatment population includes patients with SIRS and septic shock.
  • further optimization of the BRECS-d design will include the following: (1) implementation of an oxygen probe/sensor system; (2) inclusion of a filtration system design that may use miniaturized pumps or eliminate immunoprotection methods; and (3) optimization of seeding methods/cell density by way of dynamic seeding and multiple seeding.
  • a compressed BRECS-d unit is contemplated that would be wearable for therapy. This would be useful in clinical applications.
  • the BRECS-d device of Figures 13 and 14, with its cryopreservable and thawable disks, is useful to facilitate easy manufacturing and quick development at a treatment center.
  • the entire device might be cryopreserved, rather than just the disks, to further facilitate manufacturing and deployment.
  • An example of such a fully-freezable device is shown in Figures 15 and 16. Accordingly, this example describes the design considerations and testing for development of a fully-freezable embodiment of the treatment devices of the invention, such as that shown in Figures 15 and 16.
  • "BRECS" and "BRECS-d" refer to a fully-freezable treatment device, such as that shown in Figures 15 and 16.
  • the goal is to develop a prototype design to allow the entire BRECS-d unit to be cryopreserved. Additionally, the BRECS-d system will continue to be tested in vitro to assess cryopreserved shelf life, establish protocols for manufacturing, transportation and clinical deployment.
  • the prototype development will determine the robustness of the unit design within the perceived use/lifecycle environment.
  • the lifecycle process consists of the following: 1) injection molding of the unit, followed by sterilization and packaging, 2) sterile loading of the cell disk tuning fork cassettes into the unit, 3) sterile filling of the unit with freezing medium, and 4) sealing the cell holding unit for storage. The unit will then be stored in a sterile cryo-compatible bag filled with the same freezing medium.
  • pressure ports will be opened to allow for fluid expansion, and the unit will begin a temperature step- down process, in reasonable degree increments that allow for immersion in liquid nitrogen for maintenance of maximum cell viability over an indefinite length of storage time.
  • the bagged unit is removed from its liquid nitrogen storage and placed into a bath of 37 0 C water for thawing. Once the bagged unit is temperature stabilized, the pressure ports will be closed to maintain sterility as the unit is removed from the bag, dried, aseptically wiped, and connected to the ancillary circuit for patient use.
  • the resins being contemplated for injection molding are approved for biomedical use and have data showing compatibility with EtO and gamma radiation sterilization. Additionally, these materials have been used in cryo-storage tubes, but data have not been found showing performance at extended extreme low temperatures under the exact conditions of the envisioned prototype. The issues of thermal shock, stress cracking, and impact strength will be monitored, as well as differences between the coefficient of thermal expansion of the threaded fasteners and gasket material. The gaskets and fasteners are rated for the sterilization and low temperatures. The development process will address these parameters and provide data showing the robustness of the molded device's performance within its proposed environment.
  • Testing on the device will include the following.
  • BRECS-d units will be cryopreserved for increasing times to determine if there are limits to the cryo-storage time. Units will be tested at 1 week, 1 month, 3 months, 6 months, and 12 months post cryopreservation. In vitro test parameters will include 1) lactate production, 2) oxygen and glucose consumption, 3) cell release upon re-thaw and perfusion, 4) conditioned media ability to inhibit leukocyte activation, and 5) IL-8 secretion under baseline and LPS stimulated conditions. Each unit will have pre-cryopreservation baseline parameters taken for direct comparison.
  • BRECS-d units Five BRECS-d units will be cryopreserved for each of the first 4 time point with 6 BRECS-d units being used for the 1 year time point. It is contemplated that the maintenance of cell viability post-freeze will be within an acceptable standard deviation and that the integrity of the BRECS materials will be retained post-thaw. Further testing will test the longevity of the device post- freeze. 2. Develop protocols for use in manufacturing, transportation, and clinical deployment.
  • Manufacturing standard operating procedures will be developed using human renal cells. Packaging for dry ice shipment will be determined and practice shipping runs will be held utilizing actual shipping service providers. Kits will be developed for use at the clinical site for the thawing, rinsing and circuit integration of the BRECS-d unit. Thirty units will be used to carry out these planned studies (10 each for each of the protocol areas: manufacturing, transportation, and clinical deployment). 3. Release criteria will be established. Also, parameters will be established for optimal conditions for the expansion of PPRECs on the carbon disks. Cell seeding density and protocol, collagen IV-coating concentration and method, various stir rates while in culture will be assessed and cryopreservation freezing media and protocols will all be reviewed and assessed for optimization.
  • the Starwheel carbon-based disk cultures will be established utilizing the following protocol: 8 x 10 7 PPRECs per 10 mL media will be perfused under recirculating conditions into a flow through chamber containing collagen IV-coated disks (0.5 ⁇ g/cm 2 ) to allow for the PPRECs to optimally infiltrate the center of the carbon disks. Disks are incubated at 37 0 C for 3-4 h before being placed in the Starwheel Dynamatrix cell growth system. Cells are spun at 2 rpm and in 175 mL of media. Media is changed 3 times weekly by aspirating old media through side arm and adding new media. Side arm caps are loosened a quarter turn to allow for gas exchange in incubator.
  • Disks are typically used after 2-5 weeks of culture.
  • Starwheel disk tuning forks containing 4 cell-seeded carbon disks each, are sterilely transferred to the BRECS-d system.
  • the BRECS-d unit will then either be connected to the in vitro perfusion system for culture and in vitro studies or prepared for cryopreservation.
  • Tuning fork cryopreservation protocol will be followed for the initial BRECS-d cryopreservation protocols.
  • the BRECS-d chamber will be filled sterilely with a freezing medium; care will be taken so that the fill level covers all disks but still allows for expansion during the freezing process.
  • the unit will be placed in a sterile bag designed for cryopreservation and the bag will then be filled with freezing medium and sealed such that the BRECS-d unit ports can be manipulated and caps opened for release of any pressure build-up that could develop during the cryopreservation process.
  • the BRECS-d cryo-system will then be reduced in temperature in a step-down process until transfer to liquid nitrogen.
  • the BRECS-d cryo-system will be removed from liquid nitrogen storage and placed in a 37 0 C waterbath for rapid thaw. The unit will then have the ports closed while still in the cryo-bag and will then be removed once port closure and device integrity have been visually confirmed.
  • the BRECS-d will be sterilely connected to a perfusion circuit, utilizing techniques similar to those used for peritoneal dialysis catheter connection.
  • the perfusion rate will be slowly increased until a desirable rate to sustain the nutritional and oxygen needs of the cells is achieved.
  • Lactate will be measured using a commercially available kit (Bio Vision, Inc., Mountain View, CA).
  • Glucose levels will be measured via a glucometer.
  • Oxygen consumption will be measured online with, for example, a multi-frequency phase fluorometer that utilizes a fiber optic reflection oxygen probe and a ruthenium based or porphyrin complex sensor that works by fluorescence quenching of the complex to measure the partial pressure of dissolved or gaseous oxygen.
  • Cell release will be assessed via manual hemocytometer cell counts and leukocyte activation via the DCFH assay described above.
  • a BRECS-d prototype will be designed and fabricated that can be cryopreserved and thawed. This design will allow for a BRECS-d unit that has therapeutic value. A shelf life of up to 1 year will be determined using established techniques for the cryopreservation of PPREC. These cells have been thawed after several years of cryopreservation with no noticeable negative effect.
  • a series of studies will be carried out to test the efficacy of a BRECS-d unit (post- cryopreservation) seeded with primary porcine renal epithelial cells (PPRECs) in the treatment of sepsis using a large-animal model of septic shock.
  • PPRECs primary porcine renal epithelial cells
  • a variety of hemodynamic parameters, systemic cytokines, BAL parameters, systemic myeloperoxidase (MPO), survival time, urine and ultrafiltrate NGAL levels (as an indicator of acute kidney injury (AKI)), systemic free- elastase levels, and leukocyte activation parameters will be assessed and compared in sham- and cell-treated groups. Rationale [0209] The following parameters will be measured in the large animal studies.
  • Cardiovascular parameters heart rate; systolic, diastolic, and MAP; cardiac output; systemic vascular resistance; stroke volume; renal artery blood flow; central venous pressure; pulmonary capillary wedge pressure.
  • Pulmonary parameters pulmonary artery systolic and diastolic pressures, pulmonary vascular resistance, arterial to alveolar O 2 gradient.
  • Arterial blood gases p ⁇ 2 , pCO 2 , pH, total CO 2 .
  • Inflammatory indices systemic serum levels of cytokines (IL-l ⁇ , IL-6, IL-8, IL-IO, INF- ⁇ , TNF- ⁇ ), myeloperoxidase, and elastase will be measured q 2 hours.
  • Pulmonary inflammation by BAL fluid parameters will be measured at baseline and 6 hours post-bacterial infusion: protein content (vascular leak); total cell counts with differential; IL-l ⁇ , IL-6, IL-8, IL-IO, INF- ⁇ , TNF- ⁇ , neutrophil myeloperoxidase, elastase.
  • Alveolar macrophages will be isolated from BAL fluid, and various baseline and stimulated levels of cytokines assessed after LPS challenge using methods established in the laboratory.
  • Leukocyte Activation Blood will be collected in heparinized tubes at baseline and q 1 hour post-bacteria infusion at the pre-BRECS-d hemofilter and post-BRECS-d hemofilter blood ports for the processing of leukocytes for assessment of leukocyte activation
  • Urine and ultrafiltrate will be collected q lhr for relative comparison of NGAL levels, serum creatinine and BUN will measured at baseline and at the final time point.
  • pigs After administration of anesthesia and intubation, pigs will undergo placement of dialysis catheter, arterial catheter, and Swan-Ganz thermodilution catheter (which are connected to transducers) to monitor arterial blood pressure, cardiac output, and central venous pressures.
  • a Foley catheter is placed in the urinary bladder to measure hourly urine output.
  • Doppler flow probes are placed around a renal artery and connected to a flow meter (available from Transonics Systems Inc.®, Ithaca, NY). After all access lines are in place and secured and baseline parameters and laboratory studies obtained, 200 cc of broth containing 30 x 10 10 bacteria/kg body weight of E. CoIi (serotype O6:K2:H1) is instilled into the animals' peritoneal cavities.
  • cefriaxione 100 mg/kg broad spectrum antibiotic will be administered 15 min after bacteria instillation.
  • 80 mL/kg of crystalloid and 20 mL/kg of colloid (Hetastarch) is administered.
  • Systemic heparinization is administered to maintain patency of the extracorporeal circuit.
  • conventional CVVH is initiated with a Fresenius F-40 hollow-fiber dialysis cartridge (HF-I; placed pre-BRECS-d) along with a BRECS-d unit and second F-40 cartridge (HF-2; placed post-BRECS-d), as depicted in both Figures 18A and B ⁇ i.e., both circuits will be tested).
  • HF-I Fresenius F-40 hollow-fiber dialysis cartridge
  • HF-2 placed post-BRECS-d
  • Extracorporeal blood flow is regulated at 200 mL/min to the lumen of HF- 1.
  • UF production from the ECS outlet of HF- 1 is warmed to 37 0 C via a thermal regulator and maintained at a desirable rate to sustain the nutritional and oxygen needs of the cells in the BRECS-d unit, where it passes through the cell chamber and then enters the lumen of HF-2.
  • the lumen outlet UF flow rate of HF-2 is regulated at a rate of 5 mL/min to maintain physiologic pressures, and the 20 mL/min balance of the HF-2 UF exits via ultrafiltration out the ECS UF outlet, where it is once again combined with the blood that comes from the HF- 1 lumen outlet and will then be returned to the animal.
  • a balanced electrolyte replacement solution containing bicarbonate base is infused into the blood line pre-HF- 1 on a 1:1 volume replacement basis of net UF that will exit the circuit as waste via HF-2 lumen outlet.
  • Continuous volume resuscitation with normal saline will also be employed so that replacement fluid will be increased after initiation of CVVH plus BRECS-d to provide a net balance of 150 mL/h to maintain MAP and cardiac output. All treatment groups will receive identical volume resuscitation protocols. No animals will receive vasopressor or inotropic agents. Animals will be observed until no arterial waveforms can be detected or at 12 hours post-bacterial infusion, at which time the experiment is terminated.
  • Cardiovascular parameter measurements and samples for biochemical analysis are obtained at various time intervals during the experiment.
  • a BAL will be performed for assessment of pulmonary microvascular damage and inflammation in response to SIRS.
  • Leukocytes will be isolated at baseline and hourly post- bacteria infusion for assessment of the effect of cell therapy compared to sham controls on leukocyte activation.
  • Urine and ultrafiltrate samples will be collected hourly for assessment of NGAL levels as an early indicator of acute kidney injury, and serum elastase will be measured to assess neutrophil degranulation.
  • Serum chemistries will be measured with an automated chemical analyzer. Cytokine levels will be measured with commercial ELISA assay kits reactive to porcine cytokines: IL- l ⁇ , IL-6, IL-8, IL-10, TNF- ⁇ , and IFN- ⁇ (R&D Systems).
  • BAL fluid will be obtained for cell counts and cell-type distribution, protein as a measure of vascular leak, myeloperoxidase (MPO) as an indicator of the activated state of neutrophils that have infiltrated the lung air space from the lung tissue, elastase as indicator of neutrophil degranulation and cytokine levels, including IL- l ⁇ , IL-6, IL-8, IL-10, TNF- ⁇ , and IFN- ⁇ .
  • MPO myeloperoxidase
  • elastase as indicator of neutrophil degranulation and cytokine levels, including IL- l ⁇ , IL-6, IL-8, IL-10, TNF- ⁇ , and IFN- ⁇ .
  • BAL fluid is obtained with cannulation of the right middle lobe bronchus and infusion of 60 mL of saline utilizing three aliquots of 20 mL each. Each aliquot sample is obtained by brisk injection and slow withdrawal of fluid.
  • MPO will be measured via a standard published method. Protein will be measured via a standard Bio-Rad protein assay kit II. A protocol for measuring the level of leukocyte surface expression by staining freshly isolated whole blood with a FITC-conjugated anti-porcine CDl Ib antibody has been developed. To assess leukocyte activation, leukocytes will be processed from heparinized blood which is either incubated immediately with a FITC-conjugated anti-porcine CDl Ib antibody or after a 30 minute exposure to PMA. PMA acts as a positive control/maximal stimulation condition.
  • the antibody/blood mixture is then be fixed with a Becton-Dickenson's "Facs lyse” solution and the leukocyte population will then be analyzed for activated neutrophils, monocytes and eosinophils.
  • the Becton-Dickenson's "Facs lyse” solution serves a three-fold purpose: it fixes the antibody-stained leukocytes for delayed flow cytometry analysis, it destroys the red blood cells, eliminating them from the analysis, and it separates the two granulocyte populations
  • NGAL neutrophil gelatinase-associated lipocalin
  • Relative NGAL levels will be determined by western blot using an antibody that cross reacts with porcine NGAL as indicated by the expected 25kb band on western blot. Differences in NGAL levels should reflect the degree of AKI in this animal model under sham and cell treated conditions. Serum elastase will be assessed as an indicator of peripheral blood neutrophil degranulation. Neutrophil elastase is released into the serum as neutrophils undergo degranulation. Under normal circumstances the serum elastase is inhibited by ⁇ l -antitrypsin, forming an elastase- ⁇ l-antitrypsin dimer.
  • the conversion of fluorogenic substrate to detectable fluorescence will be measured on a microplate reader using the following settings: excitation wavelength, 380 nm; emission wavelength, 460 nm.
  • Controls will include samples with no added serum (negative control) and the addition of purified human neutrophil elastase (Biomol® International, Inc.).
  • ANOVA repeated-measures analysis of variance
  • BRECS-d treated animals will have better cardiovascular indices, less pulmonary dysfunction and lung inflammation, and significantly extended survival times as compared to the sham group.
  • BRECS-d treatment will act to inhibit leukocyte activation when compared to sham group.
  • This device is illustrated in Figures 15 and 16 and, as the development described above indicates, is designed to allow the entire BRECS-d unit to be frozen or cryopreserved while maintaining cell viability and holding subcomponents and also to allow holding the subject's ultrafiltrate. Cryopreservation of the device should be viable in a liquid nitrogen vapor phase at a temperature range of negative 90 to negative 150 degrees Celsius; alternatively, the device could be stored in the liquid phase at negative 190 degrees Celsius.
  • the device should be biocompatible, maintain a seal, and withstand sterilization (e.g., use an autoclave at 115 degrees Celsius for 45 minutes or immersion of the device in ethylene oxide at 50 degrees Celsius for 2 to 3 hours) and pressure (e.g., no leaks up to 600 mg Hg). Also, there should be no cracks or leaks in the device.
  • sterilization e.g., use an autoclave at 115 degrees Celsius for 45 minutes or immersion of the device in ethylene oxide at 50 degrees Celsius for 2 to 3 hours
  • pressure e.g., no leaks up to 600 mg Hg.
  • there should be no cracks or leaks in the device Prior to clinical application of the entire device, each component of the device is sterilized using, for example, ethylene oxide, gamma radiation, and/or autoclave. Thereafter, the device is frozen for four hours at negative 20 degrees Celsius, followed by an additional four hours at negative 80 degrees Celsius, followed by a transfer to liquid nitrogen vapor or liquid phase, as desired.
  • the device can be thawed in a water bath, the DMSO may be flushed out, the device can be filled with media, and then the device can be connected to an extracorporeal circuit.
  • Flow and perfusion studies showed that the freezable BRECS-d of Figures 15 and 16 achieved the goals discussed above.
  • the device maintained uniform distributed flow throughout, which showed that the device held the subcomponents.
  • the device was shown to be sealed because there was no contamination.
  • the device was also sterilizable because there was no deformation of the device. Under freezing, the device did not crack or show any leaks.
  • the device also withstood pressure, because there were no leaks at 600 mm Hg.
  • Figure 46 shows the lactate production by live cells for pre-incubated cells and post-incubated cells after freezing the device.
  • Figure 47 shows the comparable glucose consumption rates of a BRECS-d device (such as, for example, that shown in Figure 13; "BRECS-d Average") and a freezable BRECS-d device (as shown in Figures 15 and 16; "Prototype” and “Prototype Average”).
  • the seal will be further optimized, for example, by using adhesive and gaskets with the ports (including the inlet and outlet) of the device.
  • a double gasket may also be used in place of the single gasket.
  • other possible materials can be used, such as polysteel or ethylene propylene diene monomer for the gasket and ultra high molecular weight polyethylene for the housing.
  • This example describes the development of extracorporeal circuits (such as, for example, the ones shown in Figures 19-21) using peritoneal fluid, rather than blood, using a BRECS-d device (such as, for example, the ones shown in Figures 13-16). More particularly, the research described herein is focused on developing large animal models of renal insufficiency for testing the bioartificial nephronal and ultrafiltration prototypes developed previously to assess these renal based devices in an ex vivo system as to their ability to maintain renal function as well as sustain viability over time. This will validate use of the devices in humans.
  • a chronic uremic sheep model was developed for treatment with sorbent regenerated and recycling peritoneal dialysis combined with BRECS units. This model was used to test the BRECS-d device of Figure 13 in the circuits of Figures 19 and 20. Viability of cells in BRECS units will also be evaluated.
  • the recycling PD circuit consists of two high flux polysulphone filters, whose membrane is both water- and solute-permeable, allowing for differentiated vectorial transport and metabolic and endocrine activity. Any bacterial contaminants with molecular weight larger than cut-off point of polysulphone membrane are held back and/or absorbed by the membrane, thus preventing development of peritonitis.
  • a branch circuit at a desirable flow rate to sustain the nutritional and oxygen needs of the cells was initiated to maintain BRECS viability. Immunoprotection of cultured cells in the BRECS unit is achieved concurrent with long-term functional performance as long as the filter membrane is intact.
  • the BRECS is kept in a 37 0 C temperature-controlled environment to ensure optimal functionality of the cells.
  • the first sheep was maintained on recycling peritoneal dialysis for 10 days, validating feasibility of the recycling design. [0224]
  • Various compositions of PD solution have been used with this animal model.
  • peritoneal dialysate fluid with 1.5% dextrose solution (Deflex®; code 054-20201, Na 132mEq/L, Ca 3.5mEq/L, Mg 0.5mEq/L, Cl 95mEq/L, Lactate 40mEq/L, Dextrose 1.5%, available from Fresenius Medical Care North America, Waltham, MA), 4.25% dextrose solution (Deflex®; code 054-20204, Na 132mEq/L, Ca 3.5mEq/L, Mg 0.5mEq/L, Cl 95mEq/L, Lactate 40mEq/L, Dextrose 4.25%, available from Fresenius Medical Care North America, Waltham, MA), or ExtranealTM solution (Na 132mEq/L, Ca 3.5mEq/L, Mg 0.5mEq/L, Cl 96mEq/L, Lactate 40mEq/L, Dextrose 0%, Icodextrin 7.5%
  • peritoneal dialysate fluid with 2.5% dextrose solution (Deflex®; code 054-20202, Na 132mEq/L, Ca 3.5mEq/L, Mg 0.5mEq/L, Cl 95mEq/L, Lactate 40mEq/L, Dextrose 2.5%, available from Fresenius Medical Care North America, Waltham, MA) can be used.
  • Prophylactic antibiotic was given intravenously. Data showed that combining nafcilin with gentamicin at an appropriate dose to balance the benefit of peritonitis prevention and the risk of pseudocolitis is beneficial.
  • the first sheep developed peritonitis at day 4, presenting cloudy and bloody effluent. PD solution culture showed pseudomonas growth.
  • the second sheep also caught a sepsis because of a pre-existing pneumonia and peritonitis. Thus, prevention and treatment of peritonitis in a prolonged treatment is desirable.
  • Figure 20 modifications were made to the protocols, which include redesigning the PD recycling circuit to reduce branch number, pre-connecting all tubing in the system and sterilizing it before use, prolonged antibiotic application, and asepsis manipulation. Further modifications included increasing the surface area of the dialysis filter and filtering PD from the subject pre-BRECS and post-BRECS before returning to the subject. Also set up were a standard diagnosis and treatment protocol for sheep peritonitis, which include clinical diagnosis, laboratory diagnosis, empiric antibiotics selection, and specific antibiotics treatment.
  • Figures 40 and 41 Further results of the ex vivo testing of the circuits shown in Figures 19 and 20, using the BRECS-d device of Figure 13, are depicted in Figures 40 and 41.
  • the results shown in Figures 40 and 41 combine data from tests involving both the circuit of Figure 19 and the circuit of Figure 20.
  • Figure 40 shows the LREC oxygen consumption rates of ex vivo testing of the circuits illustrated in Figures 19 and 20 compared to average in vitro testing of the device illustrated in Figure 13.
  • Figure 41 shows the same data of Figure 40 in comparison with other known data for peritonitis, IM antibiotics, and fever sepsis.
  • Sorbent system [0229] Several sham operations were performed with a sorbent delivery system from Renal Solutions, Inc. A sorbent regeneration system may be put into the circuit in parallel to the BRECS circuit. The used peritoneal dialysate will be regenerated at an interval of 4 times per day, with each regeneration lasting for about 1 hour, which is very close to inflow and drainage duration for one PD exchange in clinical practice. To ensure solute and water clearance, the speed of the infusion pump in the sorbent system will be programmed to ensure the final concentration of vital solute exactly the same as fresh dialysate.
  • the freezable BRECS-d device, of Figures 15 and 16 also can be used in a peritoneal fluid circuit, such as, for example, the circuit shown in Figure 21. This circuit can be tested in a similar manner as the circuits of Figures 19 and 20. It is contemplated that using the freezable BRECS-d device will obtain similar results.
  • Example 7 describes an uremic animal model utilizing the BRECS-d device of Figure 15 and 16 and using a continuous flow peritoneal dialysis-based extracorporeal circuit as shown in Figure 55.
  • This example describes the further design and testing of the device illustrated in part in Figures 17A and B (referred to in this Example as the "BRECS-h").
  • the research described herein discusses a miniaturized bioartificial renal cell system (BRECS) device based on hollow fiber and microcarrier technology.
  • the device can be manufactured under conditions that allow for cells grown on microcarriers to be cryopreserved and stored at clinical sites for easy deployment, allowing this technology to be used in a number of indications and settings.
  • results of the work done with the larger RAD system served as initial proof-of-principle for these miniature BRECS-h devices.
  • a BRECS-h device will be fabricated that allows for cryopreservation/thawing under commercial manufacturing/clinical use-based conditions. The effects of cryopreservation on the BRECS-h viability and functionality will be determined under in vitro conditions. Additionally, conditions will be developed to optimize cell number in the BRECS- h unit, and BRECS-h maintenance parameters will be assessed to gain insight as to optimal conditions and for sustaining cell viability and functionality under simulated therapeutic circuit conditions. This research will further optimize the BRECS-h device in a pre-clinical large animal model of SIRS and sepsis, which can then be used to validate the device, for example, for use in humans.
  • the BRECS-h design allows for a simplified therapy circuit that uses a peripheral catheter access and a single blood pump system, such as the circuit shown in Figure 22, allowing for treatment to occur in a clinical setting, such as, for example, in a hospital setting outside the ICU.
  • the BRECS-h design utilizes cells grown on microcarriers or particles which are then resuspended in either a biomatrix 3-D gel or physiologic buffer, followed by seeding into the lumens of a hollow fiber bioreactor.
  • the following method of deployment to the research/clinical site is contemplated. First, a tube of cryopreserved cells on microcarriers will either be mixed with a tube of pre-measured biomatrix or physiologic buffer. The cryopreserved cells and infusate medium would be stored at the research/clinical site or a central storage facility until needed.
  • cryopreserved cells When needed, cryopreserved cells would be thawed, mixed with the biomatrix material or buffer, and syringe-loaded into the treatment device.
  • the cryopreserved cells on microcarriers already mixed with the biomatrix material or buffer (with both frozen), can be thawed and syringe-loaded into the treatment device.
  • the cryopreserved tube could contain varying numbers of cells for dose-dependent therapy.
  • Figure 48 shows a hollow fiber, similar to the fibers to be used in the BRECS-h unit, seeded with HEP-G2 cells, an immortalized hepatocyte cell line, and grown in culture for 4 weeks. The cells appear viable, even in the central core of the fiber.
  • FIG. 49 shows growth curves of two flasks (A, B), each containing PPREC-seeded microcarriers, over a 21-day time course. As can be seen in the graph, cell densities of 2 x 10 9 cells per gram of microcarrier were achieved.
  • microcarriers can readily support 1 x 10 8 cells.
  • the volume of cell/microcarrier suspension containing 1 x 10 8 cells/0.25 g is approximately 4 mL and, when combined with 3.5 mL of alginate (one of the 3-D matrices of interest) or physiologic buffer, can be loaded into the lumen of a BRECS-h unit.
  • FIG. 22 shows a therapy circuit utilizing the BRECS-h treatment device of Figures 17A and B.
  • PPRECs were seeded on gelatin-based Cultispher® microcarrier beads (available from HyClone, Inc.). These PPREC-seeded microcarriers were then grown in spinner flask culture for 21 days with samples taken for lactate production, oxygen consumption, and cell counts every day for two weeks and every other day for the last week.
  • Figure 50 shows lactate production per gram of microcarrier per hour in culture over the first 12 days in culture, with Figure 51 displaying oxygen consumption over the first 19 days in culture. The decrease in lactate production and oxygen consumption at day 8 in culture may be indicative of nutrient and oxygen limitations to support the exponential growth seen between days 6 and 8 in Figure 49.
  • microcarriers were seeded into mini- hollow fiber devices containing 3 single hollow fibers per device (hollow fiber dimensions, 500 ⁇ m inner diameter, 660 ⁇ m outer diameter). Units were seeded under two conditions: suspended in a three dimensional alginate matrix and in renal cell culture media. The microcarrier seeded hollow fibers were maintained in culture for 5 days and then fixed for histologic examination. Figure 52 shows a sample stained section. As can be seen, the microcarriers are contained in the hollow fibers at a loose packing density.
  • Frosty freezing container available from Nalge Nunc International, Rochester, NY
  • isopropyl alcohol for four hours at -2O 0 C and then transferred to liquid nitrogen for five days.
  • the microcarriers were then quick thawed in a 37°C water bath. Media was added drop-wise to a final volume of 1OmL.
  • the microcarriers were incubated in 15mL conical vials with 1OmL of media for 18 hours on a test tube rocker at 37°C. The microcarriers were then spun down, supernatant was removed, followed by trypsin digestion and trypan blue cell count to assess percent recovery vs. pre-thaw cell number estimates per vial and percent viability.
  • Figure 53 shows a comparison of the microcarriers before and after cryopreservation in the three cryopreservation solutions.
  • the microcarriers are visibly more sheared in appearance around the periphery post-cryopreservation than in the fresh, non- cryopreserved sample. Additionally, free cells can be seen in the post-cryopreserved fields.
  • Table 2 summarizes the results from the three different cryopreservation solutions, with respect to percent recovery post-thaw of a reconstituted vial of cell/microcarriers vs. the estimated pre-thaw cell number per frozen vial of cell/microcarriers, the percent viable vs.
  • the data show beneficial results from the cryo -protective agent, CryoStorTM CS5.
  • the "% Post- Thaw Recovery of Estimated Pre-Thaw Cell Number” column shows the percent ratio of the number of cells that were frozen as assessed by trypsinizing a sample of the beads aliquoted in freezing solution and staining with trypan blue in comparison to the number counted when trypsinizing a sample of the beads under each condition post-thaw and staining with trypan blue.
  • the number of both viable and non- viable cells are included in the cell count.
  • the "% Death of Post- Thaw Recovery” column shows the percent ratio of the post- thaw dispersed cells that, when counted, are not viable based on trypan blue exclusion assay.
  • the "% Viable Cells of Post-Thaw Recovered” column shows the percent ratio of post-thaw viable cells as a fraction of the total number of cells determined by cell dispersion from the microcarriers followed by cell counts (the data from the "% Post- Thaw Recovery of Estimated Pre-Thaw Cell Number” column), rather than the pre-thaw seeded cell number per gram of microcarrier.
  • the "% Viable Cells of Estimated Pre-Freeze Cell Number” column shows the percent ratio of post-thaw viable cells as a function of the total number of cells prior to freezing, where total cells are the number of cells seeded per gram of microcarrier prior to the freeze/thaw process.
  • the CS5 condition had 100% recovery of cells with almost 80% viability.
  • Table 2 Table showing the results of freeze-thaw effects for various freezing media. The overall percent recovery is highest for CryoStorTM CS5, however cell viability is better for 5% DMSO.
  • Renal epithelial cells secrete low, but measureable levels of IL-8 under baseline conditions with significant increases post-LPS stimulation.
  • Figure 54 shows IL-8 secretion rates of PPRECs grown to confluence on culture plates under baseline and 24 hours LPS stimulated conditions.
  • the baseline IL-8 secretion level was approved by the FDA to be used as a release criteria for the renal assist device in the Phase I/II and Phase II clinical trials discussed above in Example 2 (under "Clinical Evaluation of Efficacy of Renal Cell Therapy"), in which the human renal cells used in the RAD display the same characteristics and are isolated using similar protocols as the Porcine cells used in the BRECS-h.
  • the BRECS-h design allows for the device to be both miniaturized and the cells to be cryopreserved, followed by a quick thaw process and syringe loading, thus eliminating the shortcomings of the RAD design.
  • PPRECs will be seeded and expanded on microcarrier beads. Optimal cryopreservation/thaw conditions will be established for the cell- seeded microcarrier s. Thawed microcarriers will then either be resuspended in an alginate solution or in a physiologic buffer, seeded into a BRECS-h unit and, if alginate-based, gelled under increased calcium conditions. The BRECS-h system will also be tested in vitro to assess cryopreserved shelf life of the cell- seeded microcarriers 1 week, 1 month, 3 months and 6 months cryostorage. [0248] The following parameters will be measured.
  • microcarriers examples include Cultispher® gelatin-based microcarriers
  • freezing mediums will be tested for optimal recovery, viability and cell function post-cryopreservation.
  • examples include, but are not limited to, CryostorTM CS-5, HypoThermosol®-Purge Solution (available from BioLife Solutions, Inc., Bothell, WA), 5% DMSO, 10% DMSO, VS55 (a solution containing 24.2% w/v (3.1M) dimethyl sulfoxide, 16.8% w/v (2.2M) 1,2-propanediol, and 14.0% w/v (3.1M) formamide), KYO-I (5.38M ethylene glycol, 2M DMSO, 0.1M PEG 1000, and 0.00175M PVP KlO in EuroCollins solution), and any additional buffers used for cryopreservation or vitrification of organs for storage.
  • Cell viability assessment criteria will include: cell release from the microcarrier, lactate production, oxygen and glucose consumption, cell recovery, and trypan blue exclusion pre- and post-cryopreservation.
  • Cell functional assessment will include the ability of microcarrier-conditioned media to inhibit leukocyte activation and baseline and LPS stimulated IL-8 production. Both functional criteria will be assessed pre- and post-cryopreservation. It is contemplated that the stimulation potential and IL-8 production will be maintained pre- and post-freeze, as will the other metabolic markers. Because LPS stimulation is an endpoint test, multiple samples of microcarriers from the same spinner flask culture will be assessed for the pre- and post- cryopreservation comparison. 2.
  • cryo- storage shelf life study will be initiated.
  • Cell- seeded microcarriers will be cryopreserved for 1 week, 1 month, 3 months and 6 months, with the established viability and functional criteria assessed pre- and post-cryopreservation.
  • One consideration is the choice of cryoprotective agent.
  • Cryoprotective agents such as glycerol and DMSO, which increase osmolarity, effectively dehydrate cells during the freezing process and thereby eliminate damaging ice crystal formation, will be considered for optimization.
  • cells in solid organs, or in this case adhered to solid microcarrier structures do not adjust as readily as cells in suspension to the abrupt changes in osmolarity inherent to standard cryopreservation protocols.
  • microcarrier cultures have a high cell density, they should be transitioned quickly to avoid nutrient depletion; however, the diffusion of cryoprotectants is variable across solid structures and should be controlled to avoid mechanical stress to the surface cells while being allowed to permeate the microcarrier interior. A balance should be reached between these two factors.
  • Several methods will be tested to optimize both the number of cells per microcarrier and the loading capacity of microcarriers into the BRECS-h unit.
  • Microcarrier cultures seeded and maintained under various conditions will be evaluated over time to obtain growth curves in culture to assess the number of cells per gram of microcarrier.
  • the determined optimal growth parameters will then be used to test several loading protocols into the BRECS-h unit to develop a method that allows for maximal microcarriers per BRECS-h.
  • the following parameters will be measured. 1. PPRECs will be seeded and expanded on microcarrier beads. A variety of seeding techniques will be employed to maximize cell number per gram of microcarrier.
  • Cartridge orientation (1 -horizontal vs. 2-vertical), in addition to loading direction (3- against or 4-with gravity), 5-pressure loading via clamping of the luminal exit port with careful monitoring of pressure build-up, and 6-multiple seedings alternated between centrifugations will be employed to optimize the microcarrier packing density.
  • Conditions for flow rates, pressure gradients (both across the ECS to lumen path and along the pre- to post-ECS path), luminal buffer or alginate monomer composition, fiber length, and fiber density in the ECS housing will be assessed to determine which parameters support optimal oxygen and nutrient gradients to the cells grown on microcarriers and contained in the hollow fibers of the BRECS-h unit.
  • Conditions will be simulated that will allow for the BRECS-h device to be assessed with respect to viability and functionality under parameters seen in an ex vivo extracorporeal circuit, such as the circuit of Figure 22, over an extended period of time.
  • the optimal BRECS-h microcarrier packing density, discussed above, will be tested in vitro under various conditions that assess cartridge and circuit parameters.
  • heparinized bovine blood will be used in the in vitro test circuit for some of the studies in this component of the research plan. Functional assessment criteria will include lactate production, oxygen depletion, glucose consumption rates, histologic assessment, leukocyte conditioned media assay and IL-8 baseline and LPS stimulated secretion rates. Many of these studies (luminal buffer or alginate monomer composition, fiber composition, fiber outer diameter and wall thickness) will initially be performed in mini- hollow fiber units to test optimal parameters. Once these have been determined, then the BRECS-h units will be used for testing final parameters for flow rates, pressure gradients and fiber packing density. 8 BRECS-h units will be tested for each of these parameters with 8
  • the cells in the BRECS-h will prove both viable and functional with respect to the assessment test parameters.
  • Conditions for flow through the BRECS-h ECS will allow for adequate nutrient and oxygen delivery over the anticipated therapeutic application time period.
  • Hollow fiber length and/or hollow fiber packing density inside the ECS may be varied.
  • a membrane oxygenator may be used in the pre-BRECS-h section of a circuit to enhance further delivering oxygen to the cells.
  • Such an optimized BRECS-h unit may be tested in a pre-clinical large animal model of sepsis and SIRS that would target the septic shock and SIRS patient population. This model would confirm the utility of the BRECS-h and validate its use in humans.
  • This example describes a protocol for coating substrates according to the invention, such as the Nb-coated carbon disks of Figures 13-16, with collagen IV, seeding such substrates with live cells, and post-seeding culture of these seeded substrates.
  • the technique for passive coupling of proteins to the Dynamatrix carbon Nb disk matrices recommended by Dynamatrix, the manufacturer, is a one-sided static coating technique.
  • the protocol from the manufacturer calls for disks to be coated by placing full tuning fork assemblies, with four disk matrices, each into tissue culture plates and loading 150 ⁇ l of protein solution onto each individual disk.
  • a concentration of lO ⁇ g/mL is recommended by the manufacturer for the protein solution, but the protocol states that other concentrations may be appropriate depending on the specific protein and application.
  • After the solution is added there is an incubation step of one hour at room temperature to allow for protein adsorption onto the matrices. Excess fluid is then aspirated, and the matrix units are dried at room temperature in a sterile hood for one hour prior to cell seeding.
  • Double-sided coating with force applied by a one-minute 14.84xg (300rpm) centrifuge spin cycle on each side resulted in similar collagen coating of the top of the carbon Nb disk and improved coating of the middle and bottom of the disk compared to static coating.
  • the applied centrifugal force improves the distribution of collagen solution throughout the disk and results in more uniform collagen adsorption by ensuring that collagen passes into the center of the disks where it can be adsorbed during incubation.
  • the coating of the bottom surface of the disk is uniform as a result of loading collagen to both sides, in contrast to the non-uniform coating of the bottom surface of the disk loaded on only one side.
  • Collagen solution is prepared at a concentration between about 0.75 ⁇ g and about l ⁇ g collagen IV per cm of disk matrix to be coated. Prior to coating, all disks are removed from tuning fork assemblies and placed into separate wells of a 48-well cell culture plate. After collagen solution is adjusted to a pH between 7.0 and 7.5, 75 ⁇ l of collagen solution is loaded to the top of each disk. Immediately following loading, the culture plate is spun at 300rpm for one minute. The disks are then incubated in a laminar flow hood at room temperature for one hour to allow for collagen adsorption.
  • Each disk is then turned over, and 75 ⁇ l of collagen solution is added to the second side.
  • the culture plate is again spun at 14-xg (300rpm) for one minute, followed by a one -hour incubation at room temperature to allow for adsorption.
  • the disks are then transferred to a new culture plate for cell seeding.
  • the technique for cell seeding onto the Dynamatrix carbon Nb disk matrices recommended by the manufacturer is a one-sided static seeding technique similar to the collagen coating protocol described above.
  • the protocol calls for disks to be seeded by placing full tuning fork assemblies with four disk matrices each into tissue culture plates. A 75 ⁇ l cell suspension is then added drop-wise to each individual disk. The total number of cells to be seeded onto each unit is recommended within the range 2.5xlO 5 to 5.0 xlO 5 cells, but the protocol states that other seeded cell totals may be appropriate depending on user need and experience. After the cell suspension is loaded, an incubation step of 2-4 hours in a humidified incubator allows for adhesion of cells onto the matrices.
  • the amended double-sided seeding technique resulted in an improved, more uniform distribution of cells throughout the carbon Nb disk as compared to the manufacturer' s one-sided seeding technique. Cell coverage was especially improved in the middle and on the bottom surface of the two-sided disk.
  • One-sided seeding demonstrated heavy cell coverage of the top surface of the disk, moderate coverage of the middle of the disk, and minimal coverage of the bottom surface of the disk.
  • the difference in cell coverage of the top and bottom surfaces results in disks that are sided and could affect the performance of the disks in the BRECS-d units.
  • the one-sided seeding disk shows that the renal cells are able to pass through the disk pores and attach to the interior without external applied force, but loading cells from only one side of the disk showed incomplete cell coverage.
  • Double-sided seeding produced heavy coverage of the top surface, bottom surface, and middle of the disk. Compared to one-sided seeding, cell coverage was much more uniform throughout the double-sided disk.
  • the optimized protocol for seeding renal epithelial cells onto carbon Nb disks coated with collagen IV is a double- sided static seeding technique. Prior to seeding, carbon Nb disks are coated with collagen IV by the optimized coating protocol discussed above. The coated disks are placed into separate wells of a 48-well cell culture plate for seeding.
  • a cell suspension of renal epithelial cells in a customized UltraMDCK media (available from Lonza Group Ltd., Basel, Switzerland, catalog number 2042042) is prepared with a concentration of 1 xlO 6 cells per 75 ⁇ l of media.
  • IL of UltraMDCK media is mixed with ImI of a 500X Insulin, Transferrin, Ethanolamine, and Selenium (ITES) mixture for a final concentration of 0.5X (available from Lonza Group Ltd., Basel, Switzerland, catalog number 17-8392), 3 ⁇ l Triiodothyronine (T3) of 2XlO 6 M for a final concentration of 6XlO 12 M (available from Sigma- Aldrich, Inc., St.
  • the concentrations of the additives to the UltraMDCK mixture can be of various ranges.
  • the final concentration of the ITES mixture can be of a range from about 0.0 to about 0.5X
  • the final concentration of T3 can be of a range from about 0.0M to about 6XlO 12 M
  • the final concentration of hEGF can be of a range from about 0.0M to about IXlO 8 M
  • the final concentration of RA can be of a range from about 0.0M to about IX 10 "7 M.
  • UltraMDCK media is used for renal cells.
  • other media are contemplated for other types of cells, such as other eukaryotic cells, such as other mammalian cells.
  • a 75 ⁇ l cell suspension is then added to each disk, followed by 1.5 hours of incubation at 37 0 C and 5% CO 2 to allow for adhesion of cells to the matrices.
  • Each disk is then turned over, and an additional 75 ⁇ l of cell suspension is added to the second side.
  • a second 1.5 hour incubation period at 37 0 C and 5% CO 2 is carried out to allow for cell attachment.
  • Post-Seeding Culture of Carbon Nb Disks Post-seeding culture of Dynamatrix carbon Nb disks by manufacturer protocol is performed in the Starwheel system.
  • the system consists of a media reservoir which has a Starwheel spinner connected to the cap.
  • the spinner has eight arms into which tuning fork assemblies having cell-seeded disks are placed.
  • the spinner is magnetic and spins at a set rpm when the Starwheel system is placed on a stir plate.
  • Manufacturer protocol calls for seeded tuning fork assemblies to be placed into spinner arms in a sterile environment.
  • the cap is then secured onto the media reservoir.
  • the reservoir is filled with 175mL of media through a side arm, and the system is placed on CellGro spinner plates. Finally, the two side arm caps are loosened to allow for gas exchange during culture, and the spinner plate is set to the desired rpm.
  • Culture rpm recommended by the manufacturer is 2rpm, and the protocol warns against exceeding lOrpm.
  • each of these four prepared tuning fork assemblies was cultured by four different experimental techniques: manufacturer- recommended 2rpm Starwheel, a faster 4rpm Starwheel, 25mL/min BRECS-d perfusion, and a faster 50 mL/min BRECS-d perfusion.
  • the 25 mL/min and 50 mL/min perfusion rates were chosen because they were calculated as the equivalent media delivery of 2rpm and 4rpm Starwheel culture, respectively.
  • Each technique was evaluated for maintenance of renal cells on the disks for a period of two weeks in culture by assessing the signal of fluorescent cell stain on the top surface, bottom surface, and cross- sectional middle of disks by visual inspection using a camera-microscope configuration.
  • seeded disk matrices in tuning fork assemblies are placed directly into the BRECS unit.
  • Culture in the BRECS-d is performed by the perfusion of UltraMDCK media through the unit at rate of 25 to 50 mL/min. Alternatively, a perfusion rate of between about 10 mL/min and about 200 mL/min can be used.
  • This example describes the development of a cell therapy system implemented in two large animal applications, an anephric sheep model of uremia and a porcine model of sepsis.
  • the goal was to evaluate the functionality of cell-seeded BRECS-d units in pre-clinical acute and chronic therapy applications. More particularly, the chronic uremic sheep model was used to test the BRECS-d device of Figures 15 and 16 in the circuit of Figure 55. Additionally, BRECS-d therapy was applied to an acute porcine septic pig model. This model was used to test the BRECS-d devices of Figures 13-16 in the circuits of Figures 18A and B.
  • a twenty-four hour continuous flow peritoneal dialysis (CFPD) protocol was developed using normal sheep. This protocol utilized the LREC-seeded BRECS-d device of Figure 13 in circuits as shown in Figures 19 and 20.
  • the CFPD protocol was implemented in an anephric sheep model of uremia. Female sheep weighing about 40 kg were used. In brief, two peritoneal catheters were implanted into the peritoneal cavity of each sheep one week before the start of CFPD. Normal animals were treated as described in Example 4.
  • CFPD peritoneal dialysate
  • the net change in intraperitoneal fluid volume, otherwise known as the net ultrafiltration volume, in each uremic animal was estimated to be between about 480 mL/day to about 1200 mL/day.
  • CoIi serotype O6:K2:H1
  • cefriaxione 100 mg/kg
  • a broad spectrum antibiotic 80 mL/kg
  • fluid resuscitation 80 mL/kg of crystalloid and 20 mL/kg of colloid (Hetastarch) was administered.
  • a BRECS-d unit selected from those shown in Figures 13-16, each unit embodiment was tested
  • an extracorporeal blood circuit selected from those shown in Figures 18A or B, each circuit embodiment was tested.
  • the BRECS-d unit and the circuit were chosen for each study depending upon which BRECS-d device and which circuit was available on the day of the study, and the data collected from all pairings of BRECS-d units and circuits were combined.
  • acellular BRECS-d units were used.
  • PPREC-seeded BRECS-d units were employed.
  • Continuous volume resuscitation with normal saline was employed to provide a net balance of 150 mL/h to maintain MAP and cardiac output.
  • the time to death in this porcine model of bacteremic septic shock was 10-16 hours.
  • Cardiovascular parameters were measured and samples for biochemical analysis were obtained at various time intervals during the experiment.
  • PPREC-seeded BRECS-d treated animals showed overall improvement in cardiovascular parameters, with a 27% improvement in cardiac output.
  • BRECS-d therapy increased average survival time from 6.7 hours in the non-treated sham group to 11.3 hours in the BRECS-d treated group. (Of note, 3 BRECS-d treated animals were terminated at the predetermined 12 hour end point rather than expiring due to sepsis).
  • the peritoneal dialysis treatment circuit of Figure 55 provided acceptable uremic control while maintaining cell viability and functionality in HREC-seeded BRECS-d devices in a sheep model of uremia.
  • the 24-hour CFPD model can be applied as a platform for various types of cell therapy and to PD-based wearable bioartificial kidneys.
  • the circuit of Figure 55 used in this example allows for potential cell cytokine production and metabolic components to be delivered to an animal with no interference or binding within a second hemofilter. Incorporation of a BRECS-d device into an animal model of septic shock expands therapeutic application to acute kidney injury as well. This example demonstrates a device, readily adaptable to the clinic, that expands function beyond xenographic applications.
  • Example 8 Durability, Freezing and Thawing of Cell-Seeded BRECS-d Devices
  • This example describes the durability of PPREC- and HREC-seeded BRECS-d units of Figures 13-16. Further, cryopreservation and reconstitution of a BRECS-d device of Figures 15 and 16 was tested.
  • each BRECS-d device consisted of 20 trabeculated carbon disks seeded with upwards of 10 8 PPRECs.
  • the disks were coated with collagen IV using the double-sided centrifugation coating technique (as described in Example 6), and were seeded with cells using the double- sided static seeding technique (as described in Example 6).
  • Culture in each BRECS-d unit was performed by perfusing UltraMDCK media through the unit at a rate of 50 mL/min, which provided nutrients and oxygen for maintaining cell viability. Over the course of 186 days in culture, the number of cells in the BRECS-d devices increased, and each device maintained up to 3.5 x 10 8 cells.
  • HREC-seeded BRECS-d units were analyzed by immunohistochemistry (IHC). The HREC-seeded BRECS-d units were generated by a combination of the double-sided centrifugation collagen IV coating technique and the double-sided static cell seeding technique as previously described.
  • FIG. 58 shows representative images of HRECs seeded on collagen-IV coated carbon disks after BRECS-d culture of up to 90 days. In this experiment, HREC-seeded disks were removed from BRECS-d units after 90 days in culture and rinsed three times with sterile PBS.
  • the cells were fixed in cold, freshly made 4% paraformaldehyde for 20 minutes on ice, permeated with 0.1% TritonX-100 for 10 minutes on ice, and subsequently blocked for 1 hour using 5% goat serum (# G9023 Sigma-Aldrich) and 1% NaN 3 in PBS. Following the blocking incubation, disks were washed three times with PBS, and incubated overnight at 4°C with primary antibodies. After incubation, the primary antibodies were removed, followed by a 30 minute dark exposure to fluorescent secondary antibodies and to DAPI staininig. Three blocking buffer washes were performed (two minutes each wash), which was followed by extensive PBS washing prior to visualization.
  • Figure 59 shows the primary (first column of the table) and secondary (fourth column of the table) antibodies used for the IHC studies. Samples were viewed under fluorescent optics using a Zeiss Axiovert 200 fluorescent microscope. [0286] Results from these studies indicate that HRECs cultured in BRECS-d devices maintained differentiated renal characteristics for up to 90 days. As shown in Figure 58 A, staining by DAPI showed uniform distribution of the nuclei over the entire trabeculated disk surface, indicating even distribution of the cells throughout the carbon disk.
  • ⁇ GT activity was assessed.
  • ⁇ GT which is expressed on the brush border membrane of the proximal tubule cells in the kidney, initiates the cleavage of extracellular glutathione (GSH) into its constituent amino acids, which are then transported into cells for maintenance of glutathione homeostasis.
  • GSH extracellular glutathione
  • a non-destructive, metabolic living assay was established in which the degradation rate of an exogenous supplement of GSH added to a BRECS-d unit was monitored over a 60 minute period, during which time the BRECS-d unit was perfused with 70 mL of recirculating media.
  • cryopreservation of BRECS-d units and storage options were evaluated. Liquid nitrogen vapor phase cryopreservation was assessed to determine material integrity under freezing and thawing conditions. Protocols were evaluated to investigate thermodynamic control in BRECS-d units. In these studies, BRECS-d units, as shown in Figures 15 and 16, were tested. [0289] Thawing time is relevant to cell viability and clinical implementation. The initial thaw time for an entire BRECS-d unit was over 45 minutes, and sometimes lasted up to 1.5 hours. To further establish a thawing protocol, twenty six trials were conducted to develop the protocol described below.
  • Each trial involved a single acellular device of Figures 15 and 16 being frozen and thawed under varying conditions. Thereafter, four trials were conducted using the developed protocol and the BRECS-d device of Figures 15 and 16. In these trials, BRECS-d units were filled with water and a one way valve was placed at the entrance port of each device to allow for volumetric expansion. Alternatively, sealing both the entrance and exit ports with luer caps was determined to be sufficient for acellular freezing. The units were frozen by incremental decreases in temperature from 4°C to -140 0 C over a period of four hours. After varying lengths of time in liquid nitrogen, the units were removed and immediately placed in 90 0 C water bath for 12 minutes.
  • thermocouples TC
  • Figure 62 summarizes the four studies, which were performed using the established protocol. As shown, the developed protocol produced a thawed BRECS- d device in 20 minutes while maintaining an internal temperature of no more than 39°C within the device.
  • BRECS-d units (shown in Figures 15 and 16) were cryopreserved to assess cell retention rate and cell viability parameters after thawing.
  • each BRECS- d unit was initially flushed with 60 mL of sterile phosphate buffered saline (PBS) and then filled with HTS-Purge Solution (BioLife Solutions, Inc.), a hypothermic solution that helps to prepare cells for cryopreservation.
  • HTS-Purge Solution BioLife Solutions, Inc.
  • the cryopreservation buffer CryoStor CS5 which contains 5% DMSO
  • the temperature drop in each unit was conducted slowly by placement in a Styrofoam cooler and was adjusted from 4°C, to - 20 0 C, to -80 0 C, and then to the vapor phase of liquid nitrogen (-14O 0 C) over a period of four hours.
  • the units were then stored in the liquid nitrogen vapor phase for varying lengths of time.
  • PPREC-seeded BRECS-d units were thawed according to the above-described protocol for acellular devices, resulting in a transition from -140 0 C in the liquid nitrogen vapor phase to a 37°C operating temperature within 20 minutes.
  • the central core of each BRECS-d unit, which contained the cell-seeded disks, remained frozen during the initial 12 minute incubation at 9O 0 C.
  • the cells were subsequently thawed by perfusion with 37 0 C media, which rapidly replaced the cryopreservation buffer.
  • Points A and B indicate cell numbers before freezing and after thawing, respectively, for units transferred from liquid nitrogen storage to -80 0 C storage.
  • Points C and D show the cell numbers before freezing and after thawing, respectively, for units stored only in liquid nitrogen vapor phase. As shown, the two experimental groups exhibited comparable post-thaw cell retention rates.
  • thermocouples placed inside a cryopreserved acellular BRECS-d unit of Figures 15 and 16 indicates that a 36 hour transfer time is feasible as an internal temperature of -80 0 C within the shipped device is maintained during that time period ( Figure 65). It is contemplated that use of device insulation and extruded polystyrene foam for a casing is expected to extend these conditions.

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Abstract

L'invention porte sur des dispositifs thérapeutiques à base de cellules extracorporelles et sur des systèmes d'administration qui offrent une méthode d’administration thérapeutique de molécules biologiquement actives produites par des cellules vivantes en réponse à un environnement physiologique dynamique. Un mode de réalisation fait intervenir de longues fibres creuses dans lesquelles une couche de cellule se développe dans le volume intraluminal ou dans une chambre à double remplissage intérieur. Un autre mode de réalisation fait intervenir une galette ou une série de galettes permettant l’obtention d’un substrat sur lequel les cellules se développent. La ou les galettes sont introduites dans un dispositif. Le dispositif peut administrer une molécule présélectionnée, par exemple une hormone, dans la circulation systémique d'un mammifère et/ou peut administrer un élément constitué de différents produits cellulaires. Le dispositif est apte à fixer des cellules viables qui produisent et sécrètent la molécule présélectionnée dans le sang ou dans un fluide. L'invention porte également sur une méthode à invasion minimale destinée à l'introduction par voie percutanée du dispositif de l'invention dans un vaisseau sanguin ou une cavité corporelle présélectionnés.
PCT/US2009/053516 2008-08-15 2009-08-12 Dispositif thérapeutique à base de cellules extracorporelles et système d'administration WO2010019643A2 (fr)

Priority Applications (3)

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EP09807211.9A EP2323725A4 (fr) 2008-08-15 2009-08-12 Dispositif thérapeutique à base de cellules extracorporelles et système d'administration
CA2734200A CA2734200A1 (fr) 2008-08-15 2009-08-12 Dispositif therapeutique a base de cellules extracorporelles et systeme d'administration
US13/027,481 US20110190679A1 (en) 2008-08-15 2011-02-15 Extracorporeal cell-based therapeutic device and delivery system

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US18911908P 2008-08-15 2008-08-15
US61/189,119 2008-08-15

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WO2010019643A3 (fr) 2010-06-10
WO2010019643A9 (fr) 2010-04-22
CA2734200A1 (fr) 2010-02-18
US20110190679A1 (en) 2011-08-04
EP2323725A4 (fr) 2014-01-29

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